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THE  PHYSIOLOGICAL  ACTION  OF  NITRO- 
BENZENE VAPOR  ON  ANIMALS 


RECAP 


A  THESIS 

Presented  to  the  Faculty  of  the   Graduate  School 
OF  Cornell  University,  for  the  Degree  op 

DOCTOR  OF  PHILOSOPHY 


BY 


WALLACE  LARKIN  CHANDLER 


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[Reprint  from  the  Ck)mell  University  Experiment  Station  Memoir  20,  March,  191 9] 


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http://www.archive.org/details/physiologicalactOOchan 


THE  PHYSIOLOGICAL  ACTION  OF  NITRO- 
BENZENE VAPOR  ON  ANIMALS 


A  THESIS 

Presented  to  the   Faculty   of   the    Graduate    School 
OF  Cornell  University,  for  the  Degree  of 

DOCTOR  OF  PHILOSOPHY 


BY 

WALLACE  LARKIN  CHANDLER 


[Reprint  from  the  Cornell  University  Experiment  Station  Memoir  20,  March,  1919] 


QCV'^flN^' 


CONTENTS 

PAGE 

Introduction 412 

Review  of  literature ;  .  .  413 

Clinical  cases  of  poisoning 413 

Clinical  symptoms  of  poisoning 415 

Seat  of  action  of  the  drug; 416 

Experimental  cases  of  poisoning 417 

Resume  of  the  literature 426 

Apparatus  and  technique  of  present  experiments 427 

Apparatus 427 

Technique 428 

Obtaining  pure  nitrobenzene 428 

Aeration  of  the  fumigation  chamber 428 

Maintaining  constant  temperature ' .  .  429 

Histological  technique 429 

Descriptions  of  experiments 430 

Dog  I  (male  dachshund) 430 

Dog  II  (male) 433 

Dog  IV  (female) 434 

Dog  V  (female) 435 

Dog  VI  (female) 436 

Dog  IX  (male) 438 

Dog  X  (female) 439 

Dog  XI  (female) 441 

Dog  XVIII  (female) 443 

Rabbits  I  and  II,  and  Guinea  Pigs  I  and  II 444 

Rabbit  III  and  Guinea  Pig  III 445 

Gray  rats  (Af ms  decumanus) 446 

White  rats 446 

Cat  X 446 

Cat  XVIII 447 

Hen  V 448 

Hen  VI 449 

Hen  VII 450 

Hen  VIII  and  Rooster  III 451 

Pigeons  IV  and  V 451 

Observations  of  the  action  of  nitrobenzene  on  insects 452 

Fleas 452 

Biting  lice 452 

The  follicular  mite  (Demodex  folliculorum) 453 

Observations  of  the  action  of  nitrobenzene  on  internal  parasites 454 

Gapeworms  (Syngamtis  trachealis) 454 

Intestinal  worms 454 

Coccidia 455 

Experiment  to  determine  the  action  of  nitrobenzene  on  digestive  functions 455 

Experiments  to  determine  the  action  of  nitrobenzene  on  blood 455 

Blood  counts 455 

Spectroscopic  examination  of  the  blood  of  animals  poisoned  by  nitrobenzene 457 

Histological  examination  of  the  tissues  of  animals  poisoned  by  nitrobenzene 458 

Dogs 458 

Birds 461 

407 


408  Contents 

PAGE 

The  symptom  complex  of  nitrobenzene  poisoning 462 

Interpretation  of  the  symptoms  named 463 

Disturbance  of  digestive  functions 464 

Cerebellar  disturbances 464 

Cause  of  the  latent  period 466 

Conclusions 468 

Literature  cited 471 


THE  PHYSIOLOGICAL  ACTION  OF  NITROBENZENE  VAPOR  ON 

ANIMALS 


THE  PHYSIOLOGICAL  ACTION  OF  NITROBENZENE  VAPOR  ON 

ANIMALSi 

Wallace  Larkin  Chandler^ 

The  present  rapid  development  in  America  of  industries  which  utilize 
large  quantities  of  benzene  or  its  derivatives  in  the  manufacture  of  their 
products  has  greatly  augmented  the  importance  of  the  question  of 
industrial  poisoning.  This  is  especially  true  of  those  industries  in  which 
nitrobenzene  is  used  in  some  particular  stage  of  a  manufacturing  process. 
Because  of  the  pleasant  odor  and  the  retarded  physiological  action  of 
nitrobenzene  its  toxic  properties  are  not  generally  recognized,  with  the 
result  that  many  workmen  are  constantly  endangered  by  being  either  in 
actual  contact  with  the  liquid  or  exposed  to  its  poisonous  fumes.  Further- 
more, the  physiological  symptoms  of  nitrobenzene  poisoning  are  not  well 
understood,  altho  medical  literature  contains  a  large  number  of  reports 
of  such  poisoning;  and  undoubtedly  a  large  number  of  cases  of  industrial 
nitrobenzene-poisoning  have  been  referred  to  other  causes,  the  real  cause 
having  been  obscured  by  the  retarded  and  inconstant  action  of  this 
chemical. 

In  the  hope  of  obtaining  more  specific  data  regarding  the  physiological 
action  of  nitrobenzene,  the  initial  experiments  conducted  by  Dr.  M. 
Dresbach  and  the  writer  on  the  investigation  of  nitrobenzene  as  a  parasiti- 
cide (Chandler,  1917)^  were  continued.  The  present  researches  have 
resulted  in  findings  which,  it  is  hoped,  may  serve  to  make  clear  some  of 
the  factors  regarding  the  action  of  nitrobenzene  which  hitherto  have  not 
been  understood;  for  example,  the  cause  of  the  "latent  period,"  the 
rea.son  for  the  inconstancy  of  certain  symptoms,  and  the  specific  nervous 
centers  involved.  The  work  has  also  opened  up  new  fields  for  investiga- 
tion along  the  lines  of  neurology,  physiology,  and  biochemistry. 

The  writer  is  indebted  for  advice  and  assistance  to  the  following  mem- 
bers of  the  faculty  of  Cornell  University:  Dr.  M.  Dresbach,  of  the  Depart- 

'  From  the  Department  of  Physiology  and  Biochemistry,  Cornell  University  Medical  College,  Ithaca, 
New  York. 

'Formerly  Instructor  in  Parasitology,  New  York  State  College  of  Agriculture,  Cornell  University, 
Ithaca,  New  York.  Research  Assistant  in  Entomology,  Michigan  Agricultural  Experiment  Station, 
Ea-Ht  Lansing,  Michigan,  since  .July  1,  1918. 

'  Dates  in  parenthesis  refer  to  Literature  cited,  page  471. 

411 


412  Wallace  Larkin  Chandler 

ment  of  Physiology  and  Biochemistiy ;  Dr.  William  A.  Riley,  of  the 
Department  of  Entomology;  Dr.  E.  M.  Chamot,  of  the  Department  of 
Sanitary  Chemistry  and  Toxicology;  Dr.  H.  M.  Kingery,  of  the  Depart- 
ment of  Histology;  and  Professor  S.   H.   Gage,  of  the  Department  of 

Histology. 

INTRODUCTION 

Nitrobenzene  (mononitrobenzene,  nitrobenzol,  oil  of  mirbane,  artificial 
oil  of  bitter  almonds,  and  so  forth),  C6H5NO2,  is  a  clear,  straw-colored, 
oily  liquid  boiling  at  210.9°  C.  and  crystallizing  in  needles  at  5.7°  C.^  It 
has  the  odor  of  oil  of  bitter  almonds,  and  when  undiluted  has  a  pungent, 
unpleasant  taste.  It  is  soluble  in  all  parts  of  alcohol,  in  ether,  chloro- 
form, benzene,  oils,  and  liquid  fats,  and  to  some  extent  in  lipoids.  It 
is  but  slightly  soluble  in  water.  It  has  a  vapor  pressure  of  but  1  milli- 
meter of  mercury  at  53°  C.  It  is  combustible,  burning  in  the  open  air 
with  a  sooty,  yellowish  flame,  and  is  explosive  when  heated  to  high  tem- 
peratures such  as  would  be  obtained  by  throwing  it  on  red-hot  iron.  It 
was  first  made  by  Mitscherlich  (1834).  At  present  it  is  manufactured 
on  a  large  scale  by  ''adding  one  part  of  benzene  to  three  parts  of  a  mixture 
of  nitric  acid(sp.  gr.  1.40)  and  sulphuric  acid  (sp.  gr.  1.84),  this  mixture 
being  made  up  of  40  parts  of  the  former  to  60  parts  of  the  latter  " 
(Weaver,  1917).  It  is  used  in  the  manufacture  of  explosives  (indurite); 
in  the  manufacture  of  anilin,  which  is  used  extensively  in  the  making 
of  dyes;  in  perfuming  soaps,  lotions,  pomades,  and  other  toilet  articles; 
as  a  solvent  in  the  manufacture  of  shoe  polish,  floor  wax,  and  the  like; 
in  the  manufacture  of  flavoring  extracts  and  certam  liqueurs;  and  for 
flavoring  confections.  It  was  recommended  and  used  with  friction  as  a 
parasiticide  as  early  as  1863,^  and  has  recently  been  recommended  as  a 
fumigant  for  the  extermination  of  the  external  parasites  of  domesticated 
animals  (Moore,  1916). 

The  poisoning  effects  of  nitrobenzene  were  noted  as  early  as  1856. 
Since  that  time,  numerous  cases  of  fatal  poisoning  in  man  have  been 
reported,  and  several  experiments  on  animals  have  been  conducted  for 
the  purpose  of  studying  the  poisonous  action  of  the  chemical. 

*  Determined  experimentally.     (See  also  Landolt,  Bornstein,  and  Roth,  1912.) 

5  This  recommendation  stimulated  investigations  by  Ollivier  and  Bergeron  C1863)  and  by  Guttmann 
(1806). 


Physiological  Action  of  Nitrobenzene  Vapor  on  Animals    413 

REVIEW  OF  LITERATURE 

According  to  Letheby  (1865),  the  ancient  Greeks  were  apparently 
familiar  with  a  substance  the  physiological  action  of  which  is  similar  to 
that  of  nitrobenzene.     He  states  as  follows: 

It  is  said  that  Thrasyas,  the  father  of  botany,  was  so  skilled  in  the  preparation  of  drugs, 

that  he  knew  how  to  compound  a  poison  which  would kill  by  a  lingering  illness. 

Theophrastus  speaks  of  this  poison,  and  says  its  force  could  be  so  modified  as  to  occasion 
death  in  two,  three,  or  six  months,  or  even  at  the  end  of  a  year  or  two  years;  and  the 
writings  of  Plutarch,  Tacitus,  Quintilian  and  Livy  are  full  of  instances  of  what  seem  to  be 
the  same  kind  of  slow  and  occult  poisoning. 

However,  nitrobenzene  itself  was  unknown  to  modern  chemistry  until 
its  discovery  by  Mitscherhch  in  1834. 

Most  of  the  literature  relating  to  the  toxic  properties  of  nitrobenzene 
consists  of  clinical  reports  of  accidental  cases  of  poisoning,  and,  from  the 
standpoint  of  obtaining  data  on  the  physiological  action  of  the  drug,  is 
unreHable  for  the  reason  that  important  information  regarding  the  patient's 
"normal"  condition  is  entirely  lacking.  Also,  in  the  few  experiments 
recorded  dealing  with  its  physiological  action  on  animals,  the  drug  was 
administered  in  the  liquid  form  by  either  intravenous  injections  or  intro- 
duction into  the  stomach  by  means  of  a  tube,  and  very  seldom  by  vapor 
inhalation.  However,  a  review  of  some  of  these  cases,  both  clinical  and 
experimental,  may  serve  to  help  interpret  the  results  of  the  present  experi- 
ments.    Short  abstracts  of  a  few  of  these  are  therefore  given. 

clinical  cases  of  poisoning 
Probably  the  most  interesting  clinical  record  is  the  case  reported  by 
Grafe  and  Homberger  (1914)  of  a  man  who  worked  in  a  nitrobenzene 
factory  filling  containers  with  nitrobenzene.  After  working  in  this  capacity 
for  a  period  of  fourteen  days  the  man  began  to  show  symptoms  of  poisoning. 
These  symptoms  were  a  blue-gray  color  of  the  skin,  headache,  backache, 
stomach-ache,  and  vomiting.  He  continued  to  work  for  a  few  days  after 
the  onset  of  the  symptoms.  On  Friday,  October  27,  1905,  he  filled  a 
double  number  of  containers  (ordinarily  he  handled  1600  hters),  and  on 
Saturday  it  was  with  difficulty  that  he  continued  to  work.  On  Sunday 
he  felt  better,  went  into  a  saloon,  drank  two  glasses  of  beer,  and  started 
to  play  cards,  when  he  suddenly  became  ill.  He  went  to  see  his  father, 
but  felt  so  ill  indoors  that  he  went  outside  for  fresh  air;  immediately  on 
coming  into  the  air,  however,  he  fell  to  the  ground  unconscious  and  was 


414  Wallace  Larkin  Chandler 

carried  back  into  the  house.  A  doctor  who  was  called  gave  the  following 
report  of  the  symptoms:  skin  blue-gray;  pupils  dilated ;  resphation  retarded; 
pulse  weak  and  irregular;  patient  unconscious.  The  man  regained  con- 
sciousness on  the  following  day,  and  complamed  of  pains  in  his  head,  his 
stomach,  and  his  back.  On  July  30,  1906,  the  patient  was  found  to  have 
a  chronic  gastritis;  his  hemoglobin  was  125  per  cent  and  his  red-cell 
count  was  6,500,000.  His  intelligence  and  sense  of  perception  had  become 
dimmed.  By  July,  1908,  his  muscles  had  become  atrophied,  and  he  was 
extremely  emaciated.  His  blood  examination  showed  the  following: 
red-cell  count,  5,600,000;  hemoglobin,  80  per  cent;  lymphocytes,  32  per 
cent;  polymorphonuclear  leucocytes,  47  per  cent;  eosinophiles,  20  per 
cent.  By  October,  1909,  his  memory  had  failed;  otherwise  he  was  in 
about  the  same  condition  as  in  1908.  In  1911  he  was  asked  the  date; 
he  looked  for  a  calendar  and  said  that  he  did  not  know  whether  it  was  , 
1910  or  1911.  His  perception  of  distances  had  failed  also.  On  October  4, 
1912,  he  was  visited  by  the  doctor.  Wlien  asked  whether  he  recog- 
nized the  doctor,  he  said  that  he  had  seen  him  before,  but  where  and  when 
he  could  not  recall;  thus  he  showed  loss  of  perception'  of  both  tune  and 
space.  He  gave  correctly  the  names  of  his  children  but  could  not  remem- 
ber which  was  the  eldest.  In  March,  1913,  his  condition  was  about  the 
same.  .  If  he  wandered  some  distance  from  his  home  he  was  unable  to 
find  his  way  back.  Several  other  incidents  revealed  the  loss  of  perception 
of  time  and  space. 

Grafe  and  Homberger  beheve  that  the  type  of  psychosis  shawn  by 
this  patient  is  identical  with  Korsakoff's  syndrome,  in  which  there  is  a 
loss  of  memory  not  only  of  things  occurring  before  the  accident  but  also 
of  things  occurring  afterward.  However,  a  careful  analysis  of  the  symp- 
toms as  recorded  brings  out  the  probability  that  the  loss  of  perception 
of  time  and  space  was  the  principal  feature;  and  this,  according  to  JeUiffe 
(1913),  indicates  cerebellar  lesions. 

Taylor  (quoted  by  Adams,  1912)  reports  the  case  of  a  young  man  who 
worked  in  a  chemical  laborator3^  The  report  states  that  the  young  man 
placed  one  or  two  drops  of  nitrobenzene  on  his  tongue  in  order  to  remove 
the  odor  of  a  pipe  he  had  been  smoking.  He  repeated  this  action  one 
and  one-half  hours  later.  In  a  few  hours  he  was  seized  with  convulsions 
and  became  unconscious.  The  coma  lasted  for  about  six  hours,  but  the 
patient  died  in  about  fifteen  hours  after  regaining  conscioiJisness. 


Physiological  Action  of  Nitrobenzene  Vapok  on  Animals    415 

Another  case  of-  fatal  poisoning;  resulting;  from  a  small  amount  of  nitro- 
benzene is  reported  by  Stone  (1904).  The  report  states  that  a  strong 
man,  weighing  one  hundred  and  sixty  pounds,  had  stained  the  uppers  of 
a  pair  of  shoes  with  hquid  shoe-blacking.  He  put  the  shoes  on  before 
they  were  dry  and  spent  the  evening  in  a  cafe.  About  midnight  he  fell 
to  the  floor  unconscious,  and  he  died  a  few  hours  later. 

These  two  cases  are  interesting  from  the  fact  that  small  quantities  of 
the  drug  were  able  to  produce  death^  Absorption  in  each  case  was  prob- 
ably facilitated  by  the  presence  in  the  blood  of  some  solvent  for  nitro- 
benzene. In  the  latter  case,  the  ingestion  of  alcoholic  liquor  undoubtedly 
facihtated  the  absorption  thru  the  skin. 

On  the  other  hand,  a  number  of  cases  have  been  reported  in  which  the 
patient  recovered  after  the  ingestion  of  large  quantities  of  nitrobenzene. 
Two  interesting  examples  are  as  follows: 

Dodd  (1891)  reports  the  case  of  a  man  forty-seven  years  of  age,  who 
ingested  two  drams  of  nitrobenzene.  The  resulting  symptoms  were 
vomiting,  extreme  cyanosis,  fixed  jaws,  contracted  pupils.  The  patient 
eventually  recovered. 

Schild  (quoted  by  Adams,  1912)  reports  the  cases  of  three  gii'ls  who 
took  nitrobenzene  as  an  abortifacient.  The  approximate  amounts  taken 
were  as  follows:  one  ingested  5  mils,  and  recovered;  the  second  ingested 
16  mils,  and  recovered;  the  third  took  l(i  mils,  and  died. 

clinical  symptoms  of  poisoning 
Regarding  the  characteristic  clinical  symptoms  of  nitrobenzene  poison- 
ing, Tiirk  (quoted  by  Roth,  1913)  says:''  "  The  clinical  picture  consists 
on  the  one  hand  of  a  greater  or  less  stimulation  of  the  gastro-intestinal 
tract;  secondly,  of  changes  in  the  blood  which  consist  of  the  formation 
of  methemoglobin  and  destruction  of  erythrocytes,  with  associated  phe- 
nomena of  high-grade  cyanosis,  blue  skin  color,  later  icterus,  dyspnoea, 
etc."  Weisstein  (1892)  says  that  there  is  great  variation  in  the  symptoms. 
He  says  that  those  symptoms  which  might  be  called  characteristic  are: 

Great  dyspnoea,  livid,  cyanotic  color  of  the  skin,  and  characteristic  bitter-almond-oil  odor 
of  the  breath;  even  the  urine  may  have  this  odor.  In  addition  we  may  have  symptoms  which 
are  not  exactly  characteristic,  as  they  are  inconstant,  such  as  incoordination,  hesitating 
speech,  drov/siness,  numbness,  vomitinj^,  Cf)iivuIsioiis,  coma,  dilation  or  contraction  of  pupils, 
or  unequal  dilation,  nystaKuius,  irn^j^uhir  pulse.  Death  is  due  to  a  failure  of  respiration  and 
circulation.    In  animals  and  in  man  the  symptoms  may  appear  early  or  late,  even  days  late." 

■Translation  from  the  original  German. 


41(3  Wallace  Larkin  Chandler 

Wei^stein  states  further  that  the  lethal  dose  depends  on  the  form  m  which 
the  dose  is  taken,  the  condition  of  the  stomach  at  the  time  of  taking, 
and  other  factors.  • 

SEAT  OF  ACTION  OF  THE  DRUG 

Regarding  the  specific  tissues  acted  upon  by  nitrobenzene,  there  has 
been  much  speculation.  Roth  (1913)  writes  as  follows:^  "  The  action 
of  nitrobenzene  depends,  as  is  known,  upon  blood  changes.  Hence 
hematological  changes  have  excited  the  greatest  interest,  tho  in  differ- 
ent cases  the  findings  differ  greatly."  Some  writers  report  pronounced 
hemolysis  accompanied  by  leucocytosis,  and  those  who  have  subjected 
the  blood  of  patients  poisoned  by  nitrobenzene  to  spectroscopic  analysis 
have  for  the  most  part  found  an  absorption  band  occupying  nearly  the 
same  position  as  the  methemoglobin  band.  Some  have  regarded  this 
band  as  the  methemoglobin  band  and  have  concluded  that  methemoglobin 
is  formed  in  the  blood  in  cases  of  nitrobenzene  poisoning.  Others  claim 
that  the  band  is  distinct  from  the  methemoglobin  band  and  is  pecuhar 
to  nitrobenzene  alone.  A  discussion  of  these  findings  appears  on  a  later 
page  of  this  article.  The  following  notes  on  the  observations  of  blood 
changes  in  clinical  cases  may  be  of  interest: 

Roth  (1913)  reports  the  case  of  a  woman  who  drank  nitrobenzene  to 
produce  abortion.  Her  blood  became  dark  chocolate  in  color.  The  red- 
cell  count  dropped  from  4,662,000  to  3,840,000;  the  hemoglobin  dropped 
from  98  to  80  per  cent;  the  number  of  leucocytes  dropped  from  16,840 
to  9400;  and  the  lymphocytes  rose  from  14.4  to  22.3  per  cent.  There. 
was  no  methemoglobin  in  the  centrifugalized  blood  serum,  but  methe- 
moglobin appeared  in  the  red  corpuscles.  Hence  Roth  concludes  that 
methemoglobin  is  formed  inside  the  red  corpuscles.  He  thinks  that  the 
formation  of  methemoglobin  is  due  to  the  action  of  p-aminophenol,  formed 
from  the  nitrobenzene. 

Meyer  (1905)  reports  the  case  of  a  patient  who  took  about  a  teaspoonful 
of  nitrobenzene,  presumably  to  produce  abortion,  on  June  15.  On  the 
16th  and  17th  of  the  month  the  body  temperature  was  38°  C.  The  red- 
cell  count  dropped  to  2,180,000;  the  white-cell  count  dropped  to  5200; 
the  hemoglobin  was  54  per  cent.  There  was  no  morphological  change 
in  the  red  corpuscles. 


'  Translation  from  the  original  German. 


Physiological  Action  of  Nitrobenzene  Vapor  on  Animals    417 

Massini  (1910-11)  cites  two  cases.  The  first  is  that  of  a  man  thirty 
years  old,  a  worker  in  a  chemical  laboratory,  who  on  November  30  drank 
by  mistake  30  mils  of  nitrobenzene.  Vomiting  was  at  once  induced  by 
giving  1|  grams  of  copper  sulfate  in  50  mils  of  water.  The  color  of  the 
blood  was  dark  brown  until  December  3.  The  red-cell  count  had  dropped 
to  1,800,000  by  December  9,  but  returned  to  normal  by  January  11. 
The  white-cell  count  was  22,400  on  December  7,  and  6800  on  January  11. 
The  lymphocytes  rose  from  22.5  per  cent  on  December  2  to  41.7  per  cent 
on  December  26.  The  polymorphonuclears  dropped  from  76.8  per  cent 
on  December  11  to  54.7  per  cent  on  December  26. 

The  second  case  cited  by  Massini  is  one  of  chronic  poisoning  in  a  man 
thirty  years  old,  who  worked  in  a  room  with  nitrobenzene.  He  was  poorly 
nourished,  the  color  of  his  skin  was  blue-gray,  the  spleen  was  enlarged, 
the  urine  was  veiy  dark,  and  urobilin  was  detected  by  spectroscopic 
analysis.  The  man  was  achnitted  to  the  hospital  on  June  4.  The  red- 
cell  count  was  then  2,500,000,  but  it  had  risen  to  4,300,000  by  June  29. 
The  white-cell  count  was  6000  on  June  4,  had  risen  to  8400  by  June  8, 
and  had  dropped  to  3800  by  June  24.  A  microscopic  examination  of  the 
blood  revealed  embryonal  forms. 

Bondi  (1894)  reports  the  case  of  a  man  twenty-five  years  old,  who 
drank  a  "mandel  liqueur"  at  nine  o'clock  in  the  evening.  He  was 
admitted  to  the  hospital  at  four  o'clock  in  the  afternoon  of  the  following 
day.  The  red-cell  count  was  6,340,000,  the  white-cell  count  16,000; 
there  was  no  methemoglobin ;  the  hemoglobin  was  112  per  cent.  Only 
one  examination  was  made. 

experimental  cases  of  poisoning 

The  literature  dealing  with  experiments  on  animals  is  not  very  consider- 
able. Only  twice  have  intensive  experiments  been  carried  on,  by  Letheby 
(1865)  and  by  Filehne  (1878).  It  may  therefore  be  well  to  include  here 
a  short  abstract  of  the  literature  dealing  with  the  subject  from  the  experi- 
mental side. 

The  first  experiment  recorded  was  conducted  by  Jones  (1857).  He 
gave  one  dram  of  nitrobenzene  to  a  rabbit,  and  reports  that  the  rabbit 
was  killefl  instantly.  One-half  diani  of  nitrobenzene  in  two  drams  of 
water,  given  internally,  kille<l  a  cat  within  tw(>lve  hours.  This  writer 
giv(!s  no  reference  to  earlier  works,  nor  does  he  describe  the  symptoms 


418  Wallace  Larkin  Chandler 

of  nitrobenzene  poisoning.  He  had  been  working  on  the  toxic  properties 
of  a  commercial  oil  of  bitter  aimonds  which  was  known  to  contain  hydro- 
cyanic acid.  The  experiments  with  nitrobenzene  were  conducted  as  a 
side  issue  and  the  purity  of  the  nitrobenzene  is  doubtful. 

Casper  (1859)  was  led  to  experiment  with  nitrobenzene  because  in 
post-mortem  examinations  of  cases  of  poisoning,  the  cause  of  which  had 
been  diagnosed  as  hydrocyanic  acid,  he  often  detected  the  odor  of  nitro- 
benzene in  the  tissues.  He  mentioned  the  desirability  of  learning  whether 
this  drug  was  poisonous,  since  it  was  being  used  more  or  less  extensively 
in  the  perfuming  of  soaps,  pomades,  and  the  like,  and  stated  that  so  far 
as  he  knew  there  was  no  account  of  it  as  a  poison.  His  article  contains 
a  short  account  of  the  chemical  and  physical  properties  of  the  drug.  In 
his  experimental  work  he  gave  an  ounce  of  nitrobenzene  (purity  not 
stated)  to  a  rabbit  in  four  separate  doses  at  intervals  of  fifteen  minutes. 
Within  a  few  minutes  after  the  final  dose,  the  animal  fell  suddenly  on  its 
left  side.  Its  pupils  were  dilated.  Convulsions  occurred  which  involved 
the  entire  body,  and  within  a  few  minutes  the  animal  was  dead.  The 
body  was  allowed  to  remain  untouched  for  a  period  of  twenty-four  hours 
in  order  to  simulate  forensic  post-mortem  cases.  It  was  then  opened. 
No  odor  of  nitrobenzene  was  detected  either  externally  or  in  the  lower 
part  of  the  digestive  tract,  but  when  the  skull  was  opened  a  strong  odor 
of  nitrobenzene  was  given  off.  This  odor  was  so  pronounced  that  a  new- 
comer, who  was  wholly  unaware  of  the  experiment,  at  once  spoke  of 
"  almond  oil."  The  odor  was  detected  in  the  blood,  in  the  brain,  and  in 
other  tissues.  The  body  was  placed  in  a  cellar  for  two  weeks  and  at  the 
end  of  that  time  had  lost  but  very  little  of  the  odor.  ^ 

F.  Hoppe  (reported  by  Casper,  1859)  introduced  20  mils  of  nitro- 
benzene into  the  stomach  of  a  medium-sized  dog.  After  a  few  hours 
the  dog  appeared  stupid,  and  at  the  end  of  twelve  hours  it  was  found 
in  a  deep  coma.  Respiration  was  slow  and  the  skin  temperature  was 
lowered.  The  animal  was  killed  by  pithing  without  causing  convulsions. 
Blood  drawn  from  the  subclavian  vein  was  dark  brown  in  color,  and 
the  odor  of  nitrobenzene  was  detected  in  it.  The  same  odor  was 
detected  in  the  urine,  which  was  dark  brown,  in  the  bile,  and  in  all  the 
organs.  The  stomach  contained  a  few  drops  of  nitrobenzene,  and  the 
contents  of  the  stomach  were  strongly  alkaline.  The  blood  retained  the 
odor  of  the  drug  for  several  days.     Casper  concludes  that  these  experi- 


Physiological  Action  of  Nitrobenzene  Vapok  on  Animals     419 

ments  prove  nitrobenzene  to  be  a  poison;  and  that  a  distinction  between 
cases  of  poisoning  by  nitrobenzene  and  by  hydrocyanic  acid  can  be  readily 
made,  since  the  bitter-almond-oil  odor  due  to  hydrocyanic  acid  disappears 
within  three  or  four  days  —  the  chemical  (hydrocyanic  acid)  being 
destroyed  by  contact  with  the  tissues  —  while  the  same  odor  due  to  nitro- 
benzene will  persist  for  several  days. 

Ollivier  and  Bergeron  (1863)  were  led  to  study  the  action  of  nitrobenzene 
from  the  same  standpoint  as  was  Casper  —  that  is,  because  the  drug 
was  being  extensively  used  in  perfuming  toilet  soaps  and  in  making 
flavoring  extracts.  It  had  also  been  recommended  afid  used  with  friction 
for  the  cure  of  parasitic  affections.  These  investigatois  argued  that 
since  nitrobenzene  is  readily  converted  into  anilin  thru  the  action  of 
nascent  hydrogen,  it  is  conceivable  that  it  may  be  changed  into  anilin 
in  the  human  body,  and  anihn  is  a  poison.  They  gave  a  guinea  pig  ten 
or  twelve  drops  of  nitrobenzene.  The  animal,  after  the  initial  agitation 
and  profuse  salivation,  remained  motionless  for  some  time  and  then  began 
to  run  about  again  without  showing  the  least  signs  of  ill  effects  from  the 
drug.  To  another  guinea  pig  they  gave  approximately  three  grams. 
The  experiment  was  begun  at  2.12  o'clock,  and  at  2.40  the  animal  exhibited 
tremors  without  excessive  convulsions.  The  heart  beats  were  very  faint 
and  the  respiration  was  decidedly  labored.  At  3.10  the  animal  attempted 
to  turn  around  and  fell  on  its  right  side.  The  animal  died  a  few  minutes 
before  4  o'clock. 

In  another  experiment  Ollivier  and  Bergeron  exposed  the  muscles  of  a 
frog's  leg  and  placed  on  them  a  drop  of  nitrobenzene.  The  muscles 
remained  sensitive  to  electric  stimulations  for  a  long  time  and  did  not 
show  the  slightest  histological  changes.  The  investigators  then  exposed 
the  heart  of  a  living  frog  and  placed  a  few  drops  of  nitrobenzene  on  it 
without  obtaining  the  slightest  modifications  of  the  heart  beat. 

In  a  fifth  experiment  OlUvier  and  Bergeron  gave  a  large,  healthy  dog 
six  drams  of  nitrobenzene,  and  in  a  half  hour  an  additional  dose  of  five 
drams.  The  experiment  was  begun  at  12.40  o'clock.  The  animal  appeared 
agitated  and  secreted  saliva  profusely,  but  did  not  vomit.  Finally  it  lay 
down  in  a  dark  corner  and  remained  motionless  for  some  time.  At  1.20 
it  started  to  howl,  appeared  excited,  and  moved  its  head  convulsively. 
Its  tongue  was  hanging  out  and  its  eyes  were  wide  and  animated.  This 
condition  lasted  for  about  six  minutes,  when  the  dog  again  became  motion- 


420  Wallace  Lakkin  Chandler 

less.  At  5  o'clock  it  showed  tremors,  and  about  an  hour  later  its  hind 
extremities  were  paralyzed.  On  the  following  morning  it  was  found  dead 
and  rigid.  A  post-mortem  examination  showed  the  following:  The 
blood  was  about  normal,  the  cells  being  little  altered,  but  the  blood 
plasma  contained  fine,  oily  droplets  which  were  recognized  to  be  nitro- 
benzene. The  meninges  were  congested,  the  veins  turgid,  the  tongue 
and  mucosae  violet  in  color,  and  there  was  a  stasis  of  the  blood  in  the 
capillaries.  The  heart  was  dilated  and  filled  with  viscous  blood,  but  there 
were  no  clots.  The  "authors  claim  to  have  found  anilin  in  some  of  the 
organs  and  in  the  blood,  as  well  as  nitrobenzene.  They  state  that  they 
had  tested  the  nitrobenzene  for  the  presence  of  anilin  just  prior  to  the 
experiments  and  had  not  found  a  single  trace.  Hence  they  conclude  that 
their  original  assumption  was  correct  —  that  nitrobenzene  may  be  con- 
verted into  anilin  in  the  living  body  of  an  animal. 

In  another  experiment  these  investigators  caused  a  young  dog  to  ingest 
a  daily  dose  of  from  two  to  three  grams  of  the  drug  for  a  period  of  sixteen 
days.  They  then  killed  the  dog.  They  found  no  trace  of  anilin  in  any 
of  the  organs  except  the  spleen  and  the  liver. 

From  these  experiments  Ollivier  and  Bergeron  draw  the  following  con- 
clusions: (1)  that  death  due  to  nitrobenzene  poisoning  is  delayed  as 
compared  with  death  due  to  an  equal  dose  of  anilin;  (2)  that  nitrobenzene 
given  in  small  daily  doses  is  eliminated  in  part  as  such,  and  changed  in 
part  to  anilin,  which  accumulates  in  the  spleen  and  fiver;  (3)  that  the  drug 
is  in  time  eliminated  as  nitrobenzene  and  anilin.  and  is  not  changed  into 
picric  acid ;  (4)  that  animals  poisoned  by  nitrobenzene  die  with  symptoms 
of  asphyxiation;  (5)  that  the  symptoms  preceding  death  are  similar  to 
those  in  the  case  of  anilin  poisoning,  except  that  the  animal  exhibits 
tremors,  not  convulsions  of  the  whole  body  as  in  the  case  of  anilin  poison- 
ing; (6)  that  nitrobenzene  does  not  appear  to  cause  any  direct  alteration 
of  the  blood,  the  muscles,  the  heart,  the  nerves,  or  other  organs.^ 

In  a  further  experiment  with  nitrobenzene,  these  authors  placed  guinea 
pigs,  cats,  and  other  small  animals  under  a  bell  jar  and  introduced  air 
saturated  with  the  vapor  of  nitrobenzene,  allowing  a  small  opening  for 
ventilation.  Under  these  conditions  they  were  able  to  produce  death  in 
from  two  to  five  hours,  death  being  preceded  by  characteristic  symptoms 
such  as  staggering,  tremors,  and  paratysis  of  the  hind  legs. 

8  The  staining  of  nerve  cells  by  the  Nissl  method  was  not  developed  until  1885. 


Physiolouical  Action  of  Nitkobenzenk  Vai'oii  on  Animals    421 

Letheby  (1865)  carried  out  a  number  of  experiments  for  the  purpose  of 
studjdng  the  effects  of  nitrobenzene  on  dogs  and  cats.  The  drug  was 
invariably  administered  by  introducing  it  into  the  stomach.  The  results 
obtained  in  his  various  experiments  were  fairly  similar;  therefore  the 
following  report  (page  49  of  reference  cited),  which  is  quoted  verbatim, 
may  be  taken  as  characteristic  of  the  results  obtained  by  him: 

Experiment  2.^  January  16th,  1862,  at  half-past  three  p.  m.,  I  gave  half  a  drachm  of 
nitro-benzole  to  a  small  terrier  dog.  The  poison  was  poured  into  the  animal's  mouth;  it 
caused  discomfort,  as  if  from  the  unpleasant  taste,  and  produced  a  copious  flow  of  frothy 
saliva.  This,  however,  soon  subsided,  and  for  an  hour  there  was  no  perceptible  effect  beyond 
a  little  heaviness  of  look.  At  the  end  of  an  hour  the  animal  was  sick,  and  after  that  it  became 
sleepy.  In  another  hour  it  was  again  sick,  and  again  in  a  quarter  of  an  hour.  For  four 
hours  the  animal  lay  on  its  side  asleep,  and  then  some  water  was  given  to  it,  which  it  took 
freely;  from  that  time  till  midnight  nothing  appeared  to  be  the  matter  with  it,  and  the  next 
morning  it  seemed  to  be  quite  well,  and  ate  its  food  heartily.  It  remained  thus  all  day, 
and  was  left  at  night  apparently  well,  but  the  next  morning  at  half-past  six  o'clock  it  was 
found  upon  its  side  insensible.  The  b-^s  were  in  constant  motion,  as  if  the  animal  was 
running.  The  head  was  drawn  back,  and  the  muscles  of  the  neck  were  rigid,  as  if  in  spasm; 
the  eyes  were  open,  the  pupils  were  widely  dilated,  and  the  conjunctiva  was  insensible  to 
the  touch.  The  animal  lay  in  this  state  for  sixty-six  hours,  that  is,  nearly  three  days,  and 
then  it  died  as  if  from  exhaustion.  During  the  whole  of  this  time  the  legs  were  in  constant 
motion;  there  were  occasional  spasms,  and  then  a  sort  of  struggle  for  breath.  The  heart 
beat  in  an  irregular,  tumultuous  manner,  and  the  breathing  was  somewhat  laborious.  The 
total  time  which  elapsed  from  the  taking  of  the  poison  to  the  death  was  one  hundred  and 
four  and  a  half  hours. 

The  bod}^  was  opened  twelve  hours  after  death.  The  brain  and  its  membranes  were 
very  vascular;  there  was  no  odour  of  the  poison  in  any  part  of  the  body;  the  lungs  were 
slightly  congested;  the  heart  was  full  of  blood  on  the  right  side,  and  there  was  a  little  on  the 
left;  the  liver  was  of  a  deep  purple  colour;  the  gall-bladder  was  full  of  bile;  the  stomach  was 
nearly  empty,  it  only  contained  a  little  fluid  and  mucus;  there  was  no  sign  of  irritation.  On 
analysis  it  yielded  a  trace  of  aniline,  but  no  nitro-benzole;  and  nothing  was  found  in  the  brain. 

Letheby  divided  the  action  of  nitrobenzene  into  two  classes,  character- 
ized respectively  by  rapidly  developing  coma  and  by  slow  paralysis  and 
coma  after  a  considerable  period  of  inaction.  He  summarizes  the  symp- 
toms as  follows  (page  42  of  reference) : 

■^'hen  the  effects  were  speedily  fatal,  the  animals  were  soon  seized  with  giddine,ss  and  an 
inability  to  walk.  The  weakness  of  the  limbs  first  appeared  in  the  hind  extremities,  and  was 
manifested  by  a  difficulty  in  standing;  but  very  soon  it  extended  to  the  fore  legs,  and  then 
to  the  head  and  neck.  There  was  complete  loss  of  voluntary  power;  the  animals  lay  upon 
the  side  with  the  head  drawn  a  little  back,  and  with  the  limbs  in  constant  motion,  as  if  in  the  act 
of  trotting  or  running.  The  muscles  of  the  back  were  occasionally  fixed  in  spasm,  and  every 
now  and  then  the  animals  had  a  sort  of  epileptic  fit.  They  looked  distressed,  and  howled 
as  if  in  pain,  and  struggled  violently;  after  which  they  always  seemed  exhausted,  and  lay 
powerless.  The  pupils  were  widely  dilated,  the  action  of  the  heart  was  tumultuous  and  irreg- 
ular, and  the  breathing  was  somewhat  difficult.  For  some  time,  however,  the  animals 
retain«'d  their  consciousness,  and  gave  signs  of  intelligence  when  spoken  to;  but  suddenly, 
and  nfU'u  at  the  close  of  a  fit,  th(!y  became  comato.se,  the  eyes  remaining  open,  although  the 
Cf)njunctivii  was  insensible  to  touch,  and  the  movements  of  the  limbs  would  m-arly  cease, 
the  breathing  became  slow  and  ,s<jmewliat  stertorous,  and  the  animals  seemed  to  be  in  a 


422  Wallace  Larkin  Chandler 

deep  sleep.     This  condition  generally  lasted  until  they  died  —  the  duration  of   the  effects 
beinf  from  twenty -five  minutes  to  twelve  hours  after  the  administration  of  the  poison. 

When  the  action  of  the  poison  was  slower  there  was  often  no  -"/isible  effect  for  hours  or 
days.  At  fkst  there  was  always  a  little  discomfort  from  the  taste  of  the  oil;  but  this  soon 
subsided,  and  then  the  animals  appeared  to  be  in  perfect  health  for  a  day  or  more;  they  would 
run  about  as  lively  as  usual,  and  would  eat  their  food  heartily;  but  suddenly  there  would 
be  a  look  of  distress,  and  perhaps  an  attack  of  vomiting,  and  then  a  fit  of  epilepsy.  When 
this  had  subsided  the  animals  were  weak,  and  sometimes  they  were  paralyzed  in  the  hind 
extremities.  After  two  or  three  of  such  attacks,  the  loss  of  power  extended  to  the  fore  Hmbs, 
and  then  they  would  lie  upon  the  side  in  a  perfectly  helpless  condition;  after  which  the  prog- 
ress of  the  case  was  much  the  same  as  that  already  described,  except  that  it  was  considerably 
slower:  consciousness,  for  example,  would  be  retained  for  days  after  the  paralysis  had  set  in; 
and  although  the  animals  were  quite  imable  to  stand,  they  would  take  food  and  drink  when 
they  were  put  into  the  mouth;  in  fact  the  condition  in  which  they  lay  was  most  distressing; 
the  look  was  anxious  and  full  of  fear,  the  limbs  v/ere  in  constant  motion,  and  every  now  and 
then  there  would  be  a  violent  struggle,  as  if  the  creature  was  in  a  fit,  or  was  making  fruitless 
efforts  to  rise.  This  would  last  for  days,  and  then  there  would  be  either  a  gradual  resto- 
ration of  voluntary  power,  with  complete  recovery,  or  death  from  exhaustion.  The  time 
which  elapsed  from  the  administration  of  the  poison  to  the  coming  on  of  the  first  serious 
symptom  —  the  epileptic  fit  —  varied  from  nineteen  hours  to  seventy-two:  in  most  cases  it 
was  about  two  days,  and  the  time  of  death  ¥/as  from  four  to  nine  days. 

Letheby  explains  the  long  period  of  inaction  as  being  due  to  the  time 
required  for  the  conversion  of  nitrobenzene  into  anilin.  He  does  not 
explain  the  reason  for  the  difference  between  the  two  types  of  effects. 
Guttmann  (1866)  thinks  this  is  not  the  real  explanation  of  the  latent 
period,  for  if  an  animal  were  given  only  from  thirty  to  sixty  drops  of  intro- 
benzene  there  would  not  be  enough  in  the  body  to  form  anihn  since  nitro- 
benzene is  continuously  excreted  by  the  lungs.  Guttmann  states,  further- 
more, that  according  to  Bergmann  two  grams  of  anilin  is  not  fatal  to  a 
small  dog,  and  therefore  from  thirty  to  sixty  drops  of  nitrobenzene  could 
not  be  fatal  if  it  were  all  converted  into  anilin. 

Guttmann  carried  out  experiments  on  frogs,  rabbits,  pigeons,  and  chick- 
ens. He  states  that  in  frogs  he  obtained  paralysis  of  all  movements  and 
the  aboKtion  of  all  reflexes.  This  result  was  obtained  whether  the  drug 
was  given  by  mouth,  by  injection  under  the  skin,  or  by  exposure  of  the 
frog  to  the  vapor  under  a  bell  jar.  He  concludes  that  since  the  muscles 
reacted  to  stimuli,  the  action  of  the  drug  was  central,  in  contrast  with 
that  of  curare  and  coniin,  which  act  on  the  peripheral  nerve  structures. 
His  paralyzed  frogs  did  not  recover.  Dresbach  (Dresbach  and  Chandler, 
1917)  obtained  only  depressant  action  on  frogs,  but  in  his  experiments 
frogs  that  were  paralyzed  for  from  one  and  one-half  to  two  hours  recovered. 
Guttmann  produced  death  in  rabbits  by  placing  in  the  mouth  as  little 
as  I  mil  of  the  drag.  The  symptoms  reported  were  unsteadiness,  stagger- 
ing, loss  of  reflexes,  wide  pupils.     Death  resulted  in  each  case  in  about 


Physiological  Actiox  of  Nitrobenzene  Vapor  ox  Animals    423 

twelve  hours.  Post-mortem  examinations  showed  dark  blood,  congestion 
of  the  brain  membranes,  and  a  pronounced  odor  of  nitrobenzene  in  the 
tissues.  All  the  organs  were  normal.  (It  will  be  remembered  that  Ollivier 
and  Bergeron  were  unable  to  detect  the  odor  of  the  drug  in  the  tissues 
of  a  poisoned  animal.)  After  introducing  1  mil  of  nitrobenzene  into  the 
mouth  of  a  hen,  Guttmann  observed  that  the  bird  closed  its  eyes  and 
had  an  unsteady  gait,  but  recovered  shortly.  Later  he  gave  2,  mils  to 
the  same  bird,  and  it  quickly  became  unconscious  and  died  during  the 
night.     The  brain  was  hyperemic,  as  in  the  case  of  the  rabl)its. 

In  regard  to  the  action  of  the  vapor,  Guttmann  states  that  Charvet 
breathed  a  "  thick  vapor  "  of  nitrobenzene  for  several  hours  without  ill 
effects,  altho  he  had  seen  complete  anesthesia  and  sleep  produced  in 
a  dog  after  an  exposure  of  one  and  a  half  hours  to  the  vapor.  He  also 
says  that  Buisson  denies  that  the  vapor  has  any  narcotic  effect,  and  more- 
over that  Ollivier  and  Bergeron  Idlled  cats  and  guinea  pigs  by  exposing  them 
to  the  vapor  for  from  two  to  three  hours.  Guttman  therefore  placed 
pigeons  under  a  bell  jar  and  caused  them  to  breathe  the  vapor  of  the 
drug.  He  obsei'ved  no  effects  after  an  exposure  of  one  hour,  but  produced 
death  by  an  exposure  of  from  two  to  three  hours.  He  states  that  the 
symptoms  are  the  same  after  vapor  inhalation  as  after  subcutaneous 
injections  or  after  ingestion  of  the  liquid.  Guttmann  did  not  observe 
convulsions  in  the  animals  poisoned,  nor  did  he  have  a  very  long  latent 
period  as  described  by  Letheby.  He  could  not  explain  the  latent  period, 
but  believed  that  Letheby's  explanation  was  not  correct,  since  in  rabbits 
killed  by  ingesting  four  "grams  of  nitrobenzene  he  could  find  no  trace 
of  anilin  in  the  urine  or  in  the  organs.  He  used  the  calcium-hypochlorite 
test.  He  observes  that  Bergmann,  who  also  could  find  no  anilin  in  the 
tissues  of  the  poisoned  animals,  ascribes  the  cause  of  the  latent  period 
to  slow  absorption  of  the  drug;  but  Guttmann  points  out  that  this  theory 
is  not  in  accord  with  the  cause  of  the  rapid  action  which  is  often  produced. 
He  also  found  nitrobenzene  in  the  blood  of  rabbits  twenty-five  minutes 
after  subcutaneous  injections. 

Eulenberg  (1876)  killed  a  cat  by  exposing  it  to  the  vapor  of  nitro- 
benzene under  a  bell  jar.  He  describes  the  symptoms  as  staggering, 
stupor,  and  so  on.  He  states  that  the  action  of  the  vapor  is  more  rapid 
than  the  action  of  the  liquid.  This  contradicts  Guttmann,  who  found 
the  action  of  the  vapor  slower.     Eulenberg  could  find  no  trace  of  anihn. 


424  Wallace  Larkin  Chandler 

Filehne  (1878)  undertook  experiments  for  the  purpose  of  clearing  up 
some  of  the  questions  concerning  which  the  findings  of  other  writers 
varied  in  their  essential  details.  Those  questions  were:  What  is  the 
reason  for  the  latent  period?  Is  nitrobenzene  converted  into  hydrocyanic 
acid  in  the  body?  Is  it  converted  into  anihn  in  the  body?  What  is  the 
action  of  nitrobenzene  on  respiration  and  on  the  blood?  Filehne  also 
includes  among  his  list  of  problems  the  following:  spectroscopic  analysis 
of  the  blood  of  animals  poisoned  experimentally  by  nitrobenzene;  the 
action  of  nitrobenzene  on  blood  outside  of  the  animal;  the  action  of 
nitrobenzene  on  the  nervous  system  and  on  muscle  tissue;  and  the  thera- 
peutic principles  to  be  employed  in  treating  cases  of  nitrobenzene 
poisoning. 

Filehne  maintains  that  the  explanation  of  the  latent  period  on  the 
basis  of  time  required  for  the  absorption  of  the  drug  is  not  sufficient, 
since  it  cannot  explain  the  extremely  rapid  course  of  the  drug  in  some  cases. 
Furthermore,  Filehne  found  nitrobenzene  in  the  blood  within  twenty-five 
minutes  after  it  had  been  injected  subcutaneously ;  he  states  also  that 
animals  heavily  poisoned  very  quickly  exhale  nitrobenzene  in  sufficient 
amounts  to  perfume  large  volumes  of  water.  He  further  claims  that 
the  latent  period  does  not  depend  on  an  accumulative  action  of  the  drug, 
since  a  single  drop  introduced  dii'ectly  into  the  blood  stream  of  a  rabbit 
will  kill  the  animal  instantly.  He  thinks  that  the  rapidity  of  the  action 
depends  on  the  rate  in  which  the  nitrobenzene  passes  from  the  blood 
to  the  central  nervous  system.  In  cases  exhibiting  rapid  action  the  transfer 
takes  place  quickly  and  convulsions  result  (in  dogs),  while  if  the  transfer 
takes  place  slowly  the  action  is  retarded  and  paralysis  is  the  principal 
symptom.  In  the  frog,  Filehne  observed  only  paralysis.  He  was  able  to 
produce  rigor  mortis  also  in  the  hind  leg  of  a  frog  immediately  after 
injecting  nitrobenzene  into  the  aorta,  even  in  cases  when  the  muscles 
had  been  severed  from  their  connection  with  the  central  nervous  system 
by  cutting  the  ischiadic  plexus,  thus  showing  that  nitrobenzene  does 
exert  a  direct  action  on  the  muscle  tissue.  Filehne  argues  that  the  reason 
why  Olhvier  and  Bergeron  failed  to  show  any  action  of  the  drug  on  the 
exposed  frog  leg  was  that  the  lymph  in  which  the  muscles  are  bathed  may 
have  served  to  exclude  the  drug  from  direct  contact  with  the  muscles.  He 
writes  that  he  himself  has  observed  similar  results  when  the  muscles  were 
thus  protected. 


Physiological  Action  of  Nitrobenzene  Vapor  on  Animals    425 

Filehne  claims  that  nitrobenzene  is  not  converted  into  hydrocyanic 
acid  in  the  body,  since,  in  the  first  place,  the  blood  of  animals  poisoned 
by  hydrocyanic  acid  is  red  while  that  of  animals  poisoned  by  nitrobenzene 
is  dark  brown;  then,  too,  the  action  of  hydrocyanic  acid  on  muscle  tissue 
is  different  from  that  of  nitrobenzene,  as  Filehne  was  able  to  prove  by 
expenments  on  frogs;  furthermore,  Filehne  was  unable  to  detect  the 
slightest  trace  of  hydrocyanic  acid  in  the  blood  or  other  tissues  of  animals 
poisoned  by  nitrobenzene,  even  by  tests  so  delicate  as  to  detect  the  drug 
in  dilutions  of  0.0002  per  cent. 

Nor  will  Filehne  concede  that  nitrobenzene  is  converted  into  anilin 
in  the  body.  He  was  unable  to  find  any  trace  of  it,  as  were  also  Bergmann 
and  Guttmann.  He  shows  that  Letheby's  method  was  at  fault;  that, 
according  to  Hoffmann  and  Muspra,tt,  nitrobenzene  when  heated  vvith 
alcohoUc  potash  is  converted  into  azobenzol,  oxalic  acid,  and  anilin. 
(Letheby  apparently  used  the  phenylisocyanide  test,  whereas  Filehne 
applied  the  hypochlorite  test.) 

Regarding  the  action  of  nitrobenzene  on  the  blood,  Filehne  found 
that  in  frogs  and  mammals  the  blood  was  dark  brown  after  poisoning 
by  nitrobenzene,  except  in  the  case  of  rabbits,  which,  he  thinks,  die  before 
the  drug  can  act  on  the  blood.  He  could  find  no  morphological  changes 
in  the  blood-cells,  but  by  spectroscopic  analysis  he  found  an  absorption 
band  occupying  a  position  between  C  and  D  near  the  position  occupied 
by  the  absorption  band  of  acid  hematin.  He  called  this  band  the  nitro- 
henzol  hand.  It  is  possible  that  he  was  not  familiar  with  the  methemoglobin 
band,  since  the  formation  of  methemoglobin  was  demonstrated  only 
a  few  years  prior  to  his  experiments.  Filehne  was  unable  to  produce 
the  dark  brown  color  in  arterial  blood  by  shaking  it  directly  with  nitro- 
benzene; he  makes  no  statement  regarding  venous  blood.  By  blood-gae 
analysis  he  demonstrated  that  the  blood  of  animals  poisoned  by  nitro- 
benzene had  lost  its  ability  to  take  up  oxygen.  He  found  the  oxygen 
content  of  such  blood  to  be  less  than  1  per  cent,  as  against  the  normal 
17  per  cent,  while  the  carbon  dioxide  content  had  increased  in  both  absolute 
and  relative  amount. 

Filehne  states  that  the  toxicologists  have  placed  the  convulsion-producing 
poisons  in  two  categories:  (1)  those  that  produce  convulsions  in  both  frogs 
and  warm-blooded  animals  (specific  convulsion-producing  poisons) ;  and  (2) 
those  that  do  not  produce  convulsions  in  frogs  but  do  produce  convulsions 


426  Wallace  Larkix  Ch.\xdler 

in  warm-blooded  animals,  which  oon^-ulsions  are  of  a  secondaiy  natm*e 
(as  in  the  case  of  asph\-xia  of  the  brain  tissues)  and  not  due  to  a  dnect 
action  of  the  drug  on  the  nen'ous  system.  He  beheves  that  nitrobenzene 
does  not  belong  to  either  of  these  categories,  since  all  the  s^^nptoms  — 
nystagmus,  pupil  reactions,  and  the  duration  of  the  con^-ulsions  —  point 
t^  a  direct  action  of  the  drug  on  the  central  motor  apparatus  and  yet 
the  diTig  does  not  produce  con^iilsions  in  frogs.  He  says  (page  372  of 
reference  cited) . 

dass  die  bei  Xitrobeuzoh'ergiftung  axiftretenden  Kranipfe  nicht  seciindarer  Xatur  sind.  dass 
\-ielmehr  das;  Xitrobenzol  direct  erregend  auf  motorische  Centralapparate  der  Warmbliiter- 
wirke.  Uud  zwar  ist  diese  Erreguug  um  ?o  heftiger  je  schneller  der  Febertritt  des  Xitro- 
benzols  aui  dem  Blute  in  das  Protoplasma  der  Ganglienzelle  erfolgt. 

He  places  nitrobenzene  in  the  hst  with  alcohol,  ether,  and  the  like,  which 
exert  a  du^ect  action  on  the  central  ner\'ous  system. 

Regarding  therapeutics  in  cases  of  nitrobenzene  poisoning.  Filehne 
says  that  solvents  for  this  dnig.  such  as  alcohols,  milk,  and  oils,  are  to  be 
avoided.  He  recommends  blood  transfusions.  He  believes  that  the  use 
of  nitrobenzene  for  flavoring  foods,  m  flavoring  extracts,  and  in  alcohohc 
drinks  such  as  hqueurs.  should  be  prohibited. 

One  other  monograph  may  Ix^  mentioned,  a  paper  b^-  Zieger  (1903). 
Zieger  followed  a  method  similar  to  the  one  used  in  the  present  research, 
but  Ms  technique  was  faulty  in  several  respects  and  he  used  only  a  small 
number  of  animals  —  cats  and  rabbits.  He  says  that  nitrobenzol  acts 
on  the  bi"ain  and  respiratory  organs  and  on  the  blood.  He  concludes 
that  the  vapor  is  not  especially  toxic  when  inhaled  m  amoimts  ordinarily 
met  with,  but  that  absolution  of  nitrobenzol  from  the  skm  can  take  place 
readily  with  serious  results. 

RESUME   OF   THE   LITERATURE 

From  the  hteratme  here  reviewed  it  wiU  be  seen  that  the  following 
points  appear  to  be  fanly  well  estabhshed: 

1.  That  nitrol^enzene  exhibits  toxic  properties,  whether  it  is  mgested, 
apphed  to  the  skin,  inlialed.  or  admhiistered  by  subctitaneous  uiiection. 

2.  That  the  size  of  the  lethal  dose  is  extremely  variable. 

3.  That  the  symptoms  of  poisoning  are  inconstant. 

4.  That  an  inteiwal  of  time  (the  latent  period")  often  elapses  between 
the  admiuistration  of  the  poison  and  the  onset  of  the  s\Taptoms. 


Physiological  Action  of  Nituobknzene  Vapoii  on  Animals    427 

5.  That  nitrobenzene  is  not  necessarily  converted  in  the  body  into 
anihn,  hydrocyanic  acid,  or  any  other  substance  b(^fore  it  exei'ts  a  toxic 
action. 

6.  That  nitrobenzene  forms  methemoglobin  in  the  blood. 

The  following  points,  altho  suggested,  have  not  been  satisfactorily 
explained : 

1 .  The  exact  seat  of  action  of  nitrobenzene. 

2.  The  cause  of  the  latent  period. 

3.  The  reasons  for  the  variability  of  the  size  of  the  lethal  dose. 

4.  The  reasons  for  the  inconstancy  of  the  symptoms  of  poisoning. 

5.  The  significance  of  the  various  types  of  symptoms  oljservcd. 

APPARATUS  AND  TECHNIQUE  OF  PRESENT  EXPERIMENTS 
APPARATUS 

The  apparatus  used  in  these  experiments  was  designed  after  a  con- 
siderable period  of  experimentation  with  various  devices;  and,  since  it 
is  such  as  may  be  used  for  investigating  the  physiological  actions  of  a  great 
many  different  gases,  the  principal  parts  are  here  described  more  or  less 
in  detail. 

The  apparatus  consists  primarily  of  a  fumigation  chamber,  with  accessory 
devices  for  saturating,  dehydrating,  and  aerating  this  chamber.  In 
addition  apparatus  was  provided  for  determining  the  purity  of  the  nitro- 
benzene used,  such  as  devices  for  ascertaining  the  boiling  point,  the  freez- 
ing point,  and  so  on;  and  apparatus  for  determining  approximately  the 
amount  of  nitrobenzene  vapor  to  a  cubic  foot  of  space  within  the  fumigation 
chamber. 

.  The  fumigation  chamber  consists  of  a  galvanized  iron  tank  60  inches 
long,  40  inches  wide,  and  30  inches  deep.  These  dimensions  were  chosen 
so  that  the  tank  could  be  readily  carried  thru  doorways.  The  chamber 
has  a  capacity  of  43.75  cubic  fe(!t.  A  metal  fossa,  |  inch  wide  and  f  inch 
deep,  was  constructed  around  the  outer  top  edge,  to  receive  the  rim  of 
the  cover.  When  it  was  desirable  to  seal  the  tank,  the  cover  was  placed 
on  and  melted  paraffin  was  poured  into  the  fossa,  thus  rendering  the 
tank  air-tight.  The  cover  is  provided  with  two  glass  windows,  one  8  x  10 
inches  and  the  othc^r  12x  18  inches.  Tho  larger  window  is  removable, 
and  fits  into  a  slot  in  such  a  way  that  it  can  be  sealed  and  unsealed  r(>adily; 


428  Wallace  Larkin  Chandler 

paraffin  is  used  for  effecting  an  air-tight  seal.  It  was  thru  this  opening 
that  animals  were  introduced  into  the  tank,  thus  obviating  the  necessity 
of  removing  the  entire  cover  each  time.  A  small  glass  window  was  also 
built  into  one  side  of  the  tank,  thru  which  observations  of  the  temperature 
inside  could  be  made.  The  tank  rests  on  two  runners,  one  of  which  is 
lower  than  the  other  in  order  to  provide  a  slant  to  facilitate  drainage  of 
urine  thru  a  small  hole  in  one  corner  of  the  bottom  of  the  tank.  A  false 
removable  bottom  1  inch  in  height,  made  of  strong  wire  of  No.  2  mesh 
and  well  supported,  was  constructed  in  three  pieces,  for  ease  in  handling. 
This  false  bottom  serves  to  keep  the  animal  from  contact  with  its  excretions. 
Two  parallel  steel  supports  are  placed  across  the  width  of  the  tank  1| 
feet  above  the  bottom,  and  on  the  middle  of  these  supports  rests  a  wire 
cage  8  X  8  X  10  inches.  This  cage  serves  to  protect  a  small  fan,  which  is 
connected  to  a  motor  on  the  outside  by  a  shaft  passing  thru  a  tightly 
fitting  collar  in  one  side  of  the  tank.  This  cage  also  protected  a  triangular 
strip  of  cheesecloth  from  which  the  nitrobenzene  was  evaporated.  The 
container  into  which  the  cheesecloth  dipped  rested  on  an  aluminum 
tray  in  the  bottom  of  the  cage.  The  inside  of  the  tank  and  all  its  internal 
accessories  were  coated  with  paraffin  in  order  to  prevent  rusting. 

TECHNIQUE 

Obtaining  pure  nitrobenzene 

Practically  pure  nitrobenzene  was  obtained  by  redistilling  the  commercial 
liquid,  at  the  temperature  of  the  boiling  point  of  nitrobenzene,  until 
a  product  was  obtained  which  proved  experimentally  to  have  a  boiling 
point  and  a  freezing  point  corresponding  to  pure  nitrobenzene  (page  412). 

Aeration  of  the  fumigation  chamber 

The  tank  was  aerated  by  passing  air  saturated  with  nitrobenzene  into 
it  at  a  rate  determined  by  the  weight  of  the  animal  being  fumigated. 
The  air  was  saturated  by  passing  it,  after  dehydration,  thru  a  flask  con- 
taining nitrobenzene  and  kept  at  a  temperature  of  50°  C,  and  condensing 
it  by  passing  it  thru  a  series  of  U-tubes  containing  nitrobenzene.  The 
final  condensing  tubes  were  placed  inside  the  tank,  so  that  the  final  con- 
densing temperature  equaled  the  temperature  of  the  tank. 


Physiological  Action  of  Nitrobenzene  Vapor  on  Animals    429 

That  this  method  for  saturating  air  with  nitrobenzene  is  practical 
was  shown  experimentally  in  the  following  way:  A  glass-stoppered 
U-tube  containing  a  little  nitrobenzene  was  dehydrated  and  weighed  to 
constant  weight.  This  U-tube  was  then  placed  in  a  constant  temperature 
chamber,  which  was  also  a  desiccator,  with  the  final  condensing  tube, 
and  air  from  this  condensing  tube  was  passed  thru  the  U-tube  for  a  given 
period  of  time.  The  U-tube  was  then  reweighed,  and  the  fact  that  it 
had  neither  lost  nor  gained  in  weight  indicated  that  the  air  coming  from 
the  final  condenser  was  saturated. 

Maintaining  constant  temperature 

A  constant  temperature  ±1  degree  centigrade  was  maintained  in  the 
fumigation  chamber  by  regulating  the  temperature  of  the  room,  it  being 
found  that  a  direct  relation  existed  between  these  two  temperatures. 

Histological  technique 

In  preparing  sections  for  histological  studies  the  following  technique 
was  employed. 

The  animal  was  quickly  and  painlessly  killed  by  piercing  the  heart  with 
a  scalpel,  since  it  was  important  that  death  should  be  produced  without  the 
use  of  drugs  and  in  a  manner  which  would  produce  a  minimum  shock. 
The  body  was  opened  immediately,  a  cannula  was  connected  with  the  aorta, 
and  a  warm  (normal  body  temperature)  isotonic  saline  solution  was 
transfused  until  all  the  blood  was  washed  out.  The  saline  solution  was 
immediately  followed  by  the  warm  fixing  fluid,  which  consisted  of  4- 
per-cent  formaldehyde  in  a  saturated  aqueous  solution  of  corrosive  sub- 
limate. The  brain  and  the  cord  were  then  quickly  dissected  out,  small 
pieces  from  each  being  placed  directly  in  the  fixing  fluid  and  allowed 
to  remain  for  twenty-four  hours.  The  pieces  were  then  washed  in  run- 
ning water  for  twenty-four  hours,  after  which  they  were  carried  thru 
50-,  60-,  70-,  and  82-per-cent  alcohol.  They  were  allowed  to  remain  in 
82-per-cent  alcohol,  to  which  was  added  a  few  drops  of  5-per-cent  alcoholic 
iodine,  until  the  excess  corrosive  sublimate  had  been  removed.  The 
alcohol  was  changed  twice  a  day  for  a  time,  and  th(Mi  as  frequently  as  it 
became?  decolorized.  The  tissues  were  then  dehydrated,  cleared, 
emljcflded,  and  sectioned.     The  sections  were  cut  4  and  5  microns  thick 


430  Wallace  Larkin  Chandler 

and  were  fixed  on  slides  by  the  usual  methods.  They  werei  then  cleared 
and  carried  down  thru  the  various  grades  of  alcohol  to  water,  and  then 
stained  for  ten  minutes  in  hot  (70°  C.)  10-per-cent  methylene  blue  in 
saturated  anilin  oil  water  (Rasmussen  and  Myers,  1916).  When  taken 
from  the  staining  fluid  they  were  hurriedly  rinsed  in  a  large  volume  of 
water,  and  were  then  placed  directly  in  95-per-cent  alcohol  where  they 
were  allowed  to  remain  until  they  were  sufficiently  destained.  They  were 
then  dehydrated,  cleared  in  xylene,  and  mounted  in  balsam.  Corre- 
sponding tissues  from  each  of  the  poisoned  animals  and  from  the  controls 
were  carried  thru  the  same  fluids  and  stained  on  the  same  shdes. 

DESCRIPTIONS  OF  EXPERIMENTS 

A  large  number  of  experiments  were  carried  out.  Since,  however, 
the  symptoms  for  each  group  of  animals  were  in  general  fairly  similar, 
so  far  as  locahzing  the  action  is  concerned,  only  two  or  three  experiments 
from  each  group  in  which  the  animals  showed  typical  symptoms  are 
included  in  the  descriptions.  The  other  experiments  included  are  those 
in  which  the  symptoms  were  different  in  essential  details. 

DOG   I    (male    dachshund) 

Weight  of  dog,  16.4  kilograms. 

August  30,  1916  —  Dog  fumigated  at  26°  C.  for  a  period  of  five  and  one- 
half  hours.  ^ 

Time  when  fumigation  was  begun,  11.15  a.  m. 
Time  when  fumigation  was  finished,  4.45  p.  m. 

Observations :  After  having  become  accustomed  to  the  strangeness 
of  the  fumigation  chamber,  the  animal  lay  down  and  became  quiet. 
At  2  p.  m.  it  was  observed  that  the  dog  had  vomited,  urinated,  and 
defecated.  At  3  p.  m.  the  animal  was  seen  to  stagg'er  when  attempting 
to  walk  from  one  end  of  the  tank  to  the  other.  At  4  p.  m.  the  animal 
was  found  lying  on  its  side;  it  was  unable  to  hft  its  head;  its  respiration 
was  labored.  At  4.30  the  condition  was  about  the  same.  At  4.45  the 
animal  was  removed  from  the  tank.  The  respiration  was  slow  and  regular, 
except  for  intermittent  long,  deep  inhalations;  the  animal  was  unconscious; 

'  The  chamber  was  thoroly  dehydrated  before  the  experiment  was  begun,  but  in  this  instance  no  time 
was  allowed  for  saturation.  Ordinarily  sufficient  time  was  given  to  insure  saturation  of  tlie  chamber 
before  the  animal  was  introduced. 


Physiological  Actiox  of  Nitrobenzene  Vapor  on  Animals    431 

the  spinal  reflexes  were  apparently  gone;  the  conjunctival  reflexes  were 
present;  there  was  profuse  salivation.  The  muscles  of  the  entire  body 
were  wholly  relaxed  when  the  animal  was  removed  from  the  tank,  but 
soon  all  of  the  legs  became  extended  and  rigid.  This  condition  gradually 
passed  off  and  the  animal  showed  signs  of  recovery.  By  11.30  p.  m. 
the  dog  had  regained  consciousness  and  was  able  to  stand  on  its  feet. 
It  walked  slowly,  with  a  trembling,  staggering,  uncertain  gait  and  without 
aim. 

August  31  —  The  anim.al  was  found  to  have  greatly  improved.  It  refused 
food,  however,  and  howled  when  the  back  of  its  head  was  touched.  It 
would  press  its  head  against  the  attendant's  legs  or  other  objects,  and 
remain  thus  for  hours. 

Seytemher  1  —  The  dog  was  found  normally  active,  eating  meat  and 
drinking  water  freely.     No  other  effects  followed. 

September  22,  1916  —  Dog  fumigated  a  second  time,  this  time  at  23°  C. 

for  a  period  of  six  hours. 

Time  when  fumigation  was  begun,  11.10  a.  m. 

Time  when  fumigation  was  finished,  5.10  p.  m. 

Observations:  At  11.15  a.  m.  the  dog  was  observed  licking  its  chops. 
At  11.30  it  was  panting  at  intervals  as  if  short  of  breath.  At  12  m.  it  was 
slightly  drowsy.  At  12.05  p.  m.  it  was  unsteady,  sitting  on  its  haunches 
but  keeping  this  position  apparently  with  difficulty.  At  12.10  it  lay 
down  in  a  natural  position  and  closed  its  eyes;  it  would  open  its  eyes 
when  one  rapped  at  the  tank,  but  would  immediately  close  them  again. 
At  12.20  the  dog's  head  was  raised  and  the  breathing  was  slow  and 
labored.  At  12.30  the  animal  appeared  slightly  confused,  and  uncertain 
as  to  the  direction  of  the  sound  when  one  tapped  on  the  tank.  At  12.50 
the  animal  stood  up  when  called  and  walked  across  the  chamber,  but 
immediately  went  back  and  lay  down  again.  From  that  time  until 
5.10,  when  it  was  removed  from  the  tank,  the  animal  lay  quiet  but  alert 
as  if  sensing  some  danger.  After  the  dog  was  removed  from  the  fumigation 
chamber  fresh  vomit  was  found  in  the  tank,  which  had  probably  been 
(iinitted  while  th(;  chamber  was  being  opened.     A  copious  secretion  of 

diva  was  ol)serv(.d  and  the  animal  was  slightly  unsteady  on  its  feet. 
11   refused  water. 


432  Wallace  Larkin  Chandler 

September  26  —  The  animal  had  apparently  recovered  and  no  symptoms 
of  the  action  of  the  drug  appeared  until  on  this  day  (four  days  after  exposure 
to  the  vapor)  when  the  attendant  observed  that  the  dog  had  difficulty 
in  using  its  hind  legs.  An  examination  was  made  and  the  animal  appeared 
normal.  This  was  about  9  a.  m.  By  11  a.  m.,  however,  incoordination 
of  the  muscles  of  the  hind  legs  was  observed  and  the  animal  walked  with 
a  peculiar  sprawhng  gait.  This  condition  became  more  pronounced  as 
the  day  advanced,  and  by  3  p.  m.  it  was  almost  impossible  to  induce 
the  animal  to  walk  at  all.  Its  tendency  was  to  crawl  into  dark  corners 
and  hide.  Finally  the  animal  refused  to  remain  on  its  feet,  and  when 
placed  on  its  feet  it  would  stumble  and  fall  down  again.  However,  at 
4.30  p.  m.,  after  a  considerable  period  of  rest,  the  dog  walked  into  its 
kennel  with  a  slow,  staggering,  sprawling  gait. 

September  27  —  There  was  evidence  that  the  dog  had  thrashed  about  a 
good  deal  during  the  night  and  it  was  found  lying  prone  on  its  side  in  the 
kennel.  The  animal  appeared  conscious,  but  was  very  irritable  and 
thrashed  about  considerably.  At  3  p.  m.  pronounced  nystagmus  was 
observed;  the  left  pupil  was  dilated,  the  right  pupil  was  contracted,  and 
the  jaws  were  set.  At  4  p.  m.  the  animal  showed  a  tendency  to  remain 
on  its  left  side;  when  turned  on  its  right  side,  it  executed  a  right-to-left 
rotation,  finally  coming  to  rest  on  the  left  side.  The  animal  seemed  to  be 
conscious  and  wagged  its  tail  when  spoken  to.  The  flexor  muscles  of  the 
hind  legs  were  in  a  state  of  tense  tonic  contraction,  drawing  the  legs  up 
against  the  body.  The  animal  would  neither  eat  nor  drink.  The  anal 
temperature  was  37.8°  C. 

September  28  —  The  general  condition  of  the  animal  was  about  the  same. 
The  trunk  muscles  were  tremulous,  the  extensor  leg  muscles  contracted. 
The  legs  were  withdrawn  once  or  twice  when  touched,  but  finally  they 
failed  to  react  even  to  the  prick  of  a  pin,  so  tense  was  the  muscular,  con- 
traction. The  animal  made  swallowing  movements  and  could  work  its 
jaws  to  some  extent,  swallowing  water  when  placed  in  its  mouth. 
Nystagmus  was  not  so  pronounced.  The  anal  temperature  was  38.3°  C, 

September  29  —  The  animal  appeared  to  be  somewhat  improved. 
Nystagmus  was  decreasing.  The  dog  swallowed  milk  when  placed  in 
its  mouth.     The  flexor  muscles  were  relaxed.     Clonic  movements  of  the 


Physiological  Action  of  Nitrobenzene  Vapor  on  Animals    433 

hind  legs  were  observed.  The  anal  tonipcraturc  was  39°  C.  The  heart 
rate  was  increased  only  when  the  animal  struggled,  but  was  a  trifle 
irregular. 

September  30  —  The  animal  was  very  much  impi'oved.  It  ate  chopped 
meat  and  drank  milk,  and  could  raise  itself  a  little.  Its  head  waved  about 
in  an  uncontrollable  manner.     The  anal  temperature  was  38.5   C. 

October  1  —  Still  more  improvement  was  observed.  The  dog  could 
almost  regain  its  feet.  It  refused  water,  but  drank  milk  without  urging. 
In  the  afternoon  the  animal  was  able  to  stand  on  its  feet;  it  walked  eight 
or  ten  steps,  and  then  staggered  and  fell.  The  anal  temperature  was 
38.7°  C. 

October  2  —  The  animal  walked  fairly  well,  but  staggered  a  great  deal. 
It  ate  greedily. 

October  3  —  The  animal  had  regained  nearly  the  normal  use  of  its  legs 
and  was  found  running  about  with  other  dogs.  This  dog  finally  recovered 
entirely  and  never  developed  any  further  symptoms. 

It  is  interesting  to  note  that  this  animal  exhibited  symptoms  just  the 
reverse  of  those  descril^ed  by  Filehne  as  following  a  retarded  action  of 
the  drug  (page  424). 

DOG  II  (male) 
Weight  of  dog,  11.5  kilograms. 

September  1,  1916  —  Dog  fumigated  at  25°  C.  for  a  period  of  two  hours  and 

fifty-nine  minutes. 

Time  when  fumigation  was  begun,  12.40  p.  m. 

Time  when  fumigation  was  finished,  3.39  p.  m. 

Observations:  Soon  after  being  introduced  into  the  chamber,  the 
animal  was  observed  to  lick  its  chops;  it  panted  at  intervals;  respiration 
was  accelerated.  At  1.45  the  animal  appeared  restless,  howling  a  good 
deal;  it  appeared  to  stagger.  At  2.30  the  animal  was  unable  to  remain 
on  its  feet;  it  lay  on  its  side,  with  the  extensors  of  all  legs  in  tonic  con- 
vulsions. At  2.50  the  condition  was  about  the  same  as  at  2.30.  At  3  p.  m. 
the  dog  made  sounds  as  if  it  was  becoming  anesthetized ;  there  were  clonic 
convulsions  of  the  extensor  muscles  of  the  fore  legs,  and  occasional  clonic 
convulsions  of  the  extensors  of  the  hind  legs  followed  by  general  abdominal 


434  Wallace  Larkin  Chandler 

muscle  tremors;  respiration  was  quickened,  with  periodic  long,  deep 
inhalations;  the  dog  was  apparently  unconscious,  and  could  not  be  aroused; 
the  eyes  were  open  and  winking;  there  was  nothing  abnormal  about  the 
pupils,  and  no  nystagmus.  At  3.04  the  condition  was  about  the  same;  the 
dog  moaned  at  intervals.  At  3.24  the  respiration  was  40,  and  increased 
in  depth  with  periodic  long,  deep  inhalations  as  before.  At  3.25  there 
was  opisthotonos,  the  convulsions  lasting  for  about  one-half  minute 
and  being  followed  by  accelerated  respiration.  At  3.27  the  respiration 
was  52.  At  3.35  the  respiration  was  shallow.  At  3.39  the  animal  was 
removed  from  the  tank;  the  muscles  of  the  entire  body  were  relaxed, 
but  soon  the  leg  muscles  stiffened;  the  tongue  and  the  Ups  were  cyanotic. 
At  3.40  the  respiration  was  irregular,  gasping;  the  animal  was  given 
artificial  respiration  and  oxygen,  but  it  died  at  3.50. 

The  body  was  opened  immediately.  The  heart  blood  was  of  a  chocolate 
color;  the  lungs  were  a  dark  gray;  the  intestines  were  hyperemic;  the 
liver  and  the  spleen  were  coffee-colored. 

In  this  case  the  type  of  symptoms  described  by  Filehne  as  following 
a  rapid  action  of  the  drug  were  undoubtedly  shown. 

DOG  IV  (female) 

Weight  of  dog,  about  12  kilograms. 

September  27 ,  1916  — -Dog  fumigated  at  21.5°  C.  for  a  period  of  ten  hours. 
Time  when  fumigation  was  begun,  8.30  a.  m. 
Time  when  fumigation  was  finished,  6.30  p.  m. 

Observations:  Immediately  after  being  introduced  into  the  fumigation 
chamber  the  animal  lay  down  and  went  to  sleep.  It  scarcely  moved 
from  this  position  during  the  entire  ten  hours;  when  the  observer  tapped 
on  the  tank  the  animal  would  open  its  eyes;  when  the  tapping  was  loud 
it  raised  its  head  but  seemed  confused  and  could  not  follow  the  sound. 
At  4.30  p.  m.  the  respiration  was  observed  to  be  decidedly  increased. 
At  5  p.  m.  the  observer  tapped  loudly  on  the  tank,  and  the  animal  opened 
its  eyes  but  did  not  raise  its  head,  tho  it  appeared  normal.  When  removed 
from  the  tafik  at  6.30,  the  animal  v/as  lively,  eating  and  drinking  freely, 
and  depositing  a  great  quantity  of  apparently  normal  urine.  This  animal 
developed  no  symptoms  of  poisoning  aftoi-ward. 


Physiological  Action  of  Nitrobexzene  Vapor  on  Animals    435 

DO(i  V  (female) 
Weight  of  dog,  11.2  kilograms. 

October  26,  1916  —  Dog  fumigated  at  20°  C.  for  a  period  of  twelve  hours. 
Time  when  fumigation  was  begun,  8.30  a.  m. 
Time  when  fumigation  was  finished,  8.30  p.  m. 

Observations:  The  animal  rested  quietly  during  the  entire  course 
of  the  experiment,  and  was  removed  from  the  tank  apparently  unharmed. 
It  ate  heartily  of  roast  beef  and  showed  no  symptoms  of  poisoning  during 
the  next  three  days. 

October  29  —  The  animal  vomited  when  it  was  taken  out  of  the  kennel, 
but  no  other  sjanptoms  were  especially  noted  during  the  day. 

October  30  —  The  animal  was  found  on  its  side  and  was  unable  to  stand. 
The  following  symptoms  were  observed :  lack  of  coordination  of  the  muscles 
of  the  extremities;  neck  muscles  rigid  and  head  drawn  back  on  the  body; 
ventroflexion  of  back;  fore  legs  drawn  up  to  the  body;  one  or  both  hind  legs 
involved  in  clonic  convulsions;  nystagmus. 

October  31  —  The  condition  was  slightly  improved.  The  fore  legs  were 
drawn  up  as  before;  the  hind  legs  were  extended,  but  were  flexed  on  the 
body.  The  right  pupil  was  widely  dilated  and  the  left  pupil  was  con- 
tracted; this  is  just  the  reverse  of  the  pupillary  reactions  observed  in 
Dog  I.  Nystagmus  was  still  in  evidence.  The  neck  muscles  were  not 
so  rigid.  The  dog  was  very  restless  all  day,  but  quieted  down  toward 
evening.     It  swallowed  milk  when  placed  in  its  mouth. 

November  1  —  There  were  no  signs  of  nystagmus.  The  general  condition 
was  very  much  improved.  The  dog  swallowed  milk  when  placed  in  its 
mouth.  The  leg  muscles  were  not  particularly  involved.  Both  pupils  were 
widely  dilated.     The  animal  appeared  to  be  conscious  but  did  not  howl. 

November  2  —  The  dog's  condition  was  very  much  improved.  It  noticed 
the  ob.server  as  soon  as  he  entered  the  room.  The  animal  took  milk 
freel3^  Nystagmus  was  observed  at  times.  The  pupils  were  somewhat 
dilated.     The  animal  was  al)le  to  raise  its  head. 

November  3  —  The  condition  was  still  more  improved.  The  animal 
drank  milk  and  water  readily.  Its  head  was  raised.  Its  fore  legs  were 
f(jld(,'d  beneath  the  body,  but  were  stiff.    . 


436  Wallace  Larkin  Chandler 

November  4  —  The  animal  was  able  to  stand  but  was  unsteady  on  its 
feet.     It  ate  meat  and  drank  milk  and  water. 

November  5  —  The  condition  was  about  normal.  The  dog's  appetite  was 
good.  The  heart  and  the  respiration  were  apparently  not  ajffected  in 
this  case. 

January  20,  1917  —  Dog  fumigated  a  second  time,  this  time  at  20°  C. 

for  a  period  of  five  hours. 

Time  when  fumigation  was  begun,  2.45  p.  m. 

Time  when  fumigation  was  finished,  7.45  p.  m. 

Observations:  As  before,  the  animal  remained  quiet  during  the  entire 
course  of  the  experiment,  and  was  taken  from  the  tank  apparently 
unharmed,  and  eating  and  drinking  heartily. 

January  21 — No  symptoms  had  appeared  by  morning.  At  8  p.  m., 
however,  the  animal  exhibited  an  apparent  stiffness  in  the  hind  legs. 
This  passed  off  dining  the  night,  and  no  further  symptoms  were  developed 
until  on  the  morning  of  January  24,  when  a  loss  of  coordination  of  the 
muscles  of  the  hind  legs  was  observed  and  the  animal  walked  with  a 
sprawling  gait  very  similar  to  that  shown  by  Dog  I.  Toward  evening 
this  condition  was  much  more  pronounced,  and  it  persisted  during  the 
course  of  two  days  altho  the  animal  developed  no  further  symptoms. 

January  27  —  All  lameness  was  apparently  gone,  and  recovery  was 
complete. 

DOG  VI  (female) 
Weight  of  dog,  10.7  kilograms. 

October  27,  1916  —  Dog  fumigated  at  25°  C.  for  a  period  of  three  hours. 
Time  when  fumigation  was  begun,  3.05  p.  m. 
Time  when  fumigation  was  finished,  6.05  p.  m. 

Observations:  The  animal  remained  quiet  during  the  experiment 
and  was  removed  from  the  tank  in  a  perfectly  normal  condition.  It  ate 
heartily  of  roast  beef.     It  had  developed  no  symptoms  by  November  3. 

November  3,  1916  —  Dog  fumigated  a  second  time,  this  time  at  22°  C. 
for  a  period  of  four  hours  and  forty -five  minutes. 
Time  when  fumigation  was  begun,  3  p.  m. 
Time  when  fumigation  was  finished,  7.45  p.  m. 


Physiological  Action  of  Nitrobenzene  Vapor  on  Animals    437 

Obsei-vations :    -The  animal   was   removed   from  the  tank  apparently 
unharmed,  and  developed  no  symptoms  during  the  night. 

November  4  —  At  8  a.  m.  the  dog  was  running  about  in  a  lively  condition 
and  was  apparently  normal.  At  2  p.  m.,  however,  the  animal  was 
found  in  convulsions;  the  legs,  particularly  the  hind  legs,  exhibited 
tetany-  and  the  muscles  of  the  abdomen  quivered  violently.  By  6  p.  m. 
the  convulsions  had  become  even  more  pronounced,  and  the  animal 
refused  food  and  drink. 

November  5  —  At  10  a.  m.  the  animal  was  found  with  its  body  flexed 
to  the  left,  and  rigid;  all  the  legs  were  rigidly  extended;  when  an  attempt 
was  made  to  straighten  the  animal  out,  the  head  would  crash  violently 
against  the  floor  and  the  animal  would  immediately  return  to  its  former 
position.  At  2  p.  m.  the  animal's  condition  had  not  changed  and  it  was 
decided  to  attempt  to  relieve  its  condition  b}^  a  blood  transfusion.  At 
4  p.  m.  this  operation  was  undertaken;  the  animal  became  anesthetized 
with  ether  very  readily,  and  did  not  struggle  on  coming  out  of  the 
anesthesia;  approximately  200  mils  of  dark  coffee-colored  blood  was 
drawn  from  the  carotid  artery  of  the  poisoned  animal,  and  500  mils 
of  defibrinated  blood  from  a  healthy  dog  was  transferred  thru  the 
femoral  vein.  At  7  p.  m.  the  animal  was  found  in  a  stupor;  the  res- 
piration was  fairly  regular.  At  9  p.  m.  the  respiration  was  very  irreg- 
ular and  labored;  at  intervals  of  about  one  and  one-half  minutes  there 
appeared  incoordinated  movements  of  the  muscles  of  the  diaphragm 
and  the  chest,  each  set  working  alternately  with  the  result  that  no  air 
was  inhaled;  these  spasms  lasted  for  from  one-half  to  three-fourths  of 
a  minute,  and  at  their  height  all  the  legs  would  move  as  if  the  animal 
were  swimming,  and  would  then  become  extended  and  rigid;  the  muscles 
of  the  abdomen  would  quiver,  then  the  animal  would  give  one  or  two 
deep  gasps  and  regular  respiration  was  resumed  for  a  time  but  gradually 
became  lessened  in  depth  again  until  the  incoordinated  movements  reap- 
pciared;  an  attempt  was  made  to  obtain  a  kymographic  record  of  the 
respiration,  but  the  animal  became  so  active  that  this  was  impossible.  At 
10  p.  m.  the  animal's  condition  was  about  the  same,  tho  a  shght  improve- 
ment in  the  respiration  was  observed;  the  heart  rate  was  52,  the  res- 
piration 30-40, 


438  Wallace  Larkin  Chandler 

November  6  —  The  animal  seemed  not  to  be  greatly  improved;  the  respira- 
tion was  irregular,  with  a  tendency  to  return  to  the  type  observed  the 
day  before,  but  it  never  reached  that  type  again  sufficiently  to  give  a 
good  record      At  12  m.  the  anal  temperature  was  26.8°  C.;the  animal 
urinated  and  passed  very  dark   soft  feces;  the   external   anal   sphincter 
was  relaxed,  but  the  internal  sphincter  was  about  normal;  the  respira- 
tion was  regular  but  very  weak;   the   animal   made   swallowing   move- 
ments and  was  given  a  very   little   water,  which   was    swallowed   with 
difficulty.     At  12.30  p.  m.  the  animal   was    placed    m    a   warm   room 
(30°  C)-  the  respiration  was  fairly  regular  but  weak,  and  the  animal 
gave  occasional  gasps.     At   2.30   p.    m.    the   external   temperature   was 
rather  low  and  the  room  temperature  was  therefore  increased;  the  dogs 
respiration  was  48,  and  was  regular  but  very   shallow;   the  heart   rate 
was  64  and  was  very  regular.      At   3   p.  m.    1    milligram   of   strychnin 
sulfate  was  injected;  the  anal  temperature   was   30.5°    C,  the  external 
anal  sphincter  was  still  relaxed.     At  4  p.  m.  the  respiration  had  improved 
to  some  extent,  but  it  became  shallow  again  at  4.30;  the  rate  was  about, 
50  a  minute;  the  heart  rate  was  75.     At  5  p.  m.  the  annnal  was  found 
gasping  weakly;  the  heart  rate  was   above    100.     The   animal   died   at 
5.10,  apparently  as  the  result  of  respiratory  failure;   the  sound  of  the 
heart  indicated  that  that  organ  was  in  excellent  condition.     At  6  p.  m 
the  body  was  opened;  the  general  condition   of  the   organs   was   found 
to  be  good;  the  spleen  and  pancreas  were  of  a   dark   blue    color;   the 
peritoneum  was  shghtly   hyperemic;    the    stomach    and    the    intestines 
were  slightly  hyperemic,  with  occasional  hemorrhagic  areas  possibly  due 
to  roundworms  which  were  present  in  large  numbers;  the  rectum  contained 
a  small  amount  of  soft,  brown  feces.     There  was  no  nystagmus  observed 
in  this  case,  nor  did  the  pupils  appear  to  be  involved.  , 

DOG  IX  (male) 

Weight  of  dog,  ?.    (The  dog,  which  was  a  very  small  one,  was  not  weighed. 

Its  weight  was  probably  about  3  kilograms,  and  it  was  completely  free 

of  excess  fat.     This  dog  had  a  severe  Demodex  infection.) 

December  4,  1916  -  Dog  fumigated  at  22°  C.  for  a  period  of  five  hours. 

Time  when  fumigation  was  begun,  12.30  p.  m. 

Time  when  fumigation  was  finished,  5.30  p.  m. 


Physiological  Action  of  Nitrobenzene  Vapor  on  Animals    439 

Observations:  The  dog  appeared  restless  during  most  of  the  time  it 
was  in  the  fumigation  chamber.  At  1.30  p.  m.  it  had  vomited.  At  5 
p.  m.  it  was  observed  to  be  unsteady  on  its  feet;  it  staggered  and  fell, 
regained  its  feet,  and  fell  again.  At  5.30,  when  it  was  removed  from  the 
tank,  the  animal  was  able  to  walk  but  staggered  about  veiy  much  as  if 
it  had  been  intoxicated  with  alcohol;  it  ate  cooked  meat. 

December  o  —  The  animal  was  found  lying  on  its  side  in  a  helpless  con- 
dition; the  tongue  and  the  lips  were  cyanotic;  the  skin  temperature 
was  very  low;  the  heart  rate  was  70,  but  was  regular;  the  respiration 
was  irregular,  as  if  from  disorganization  of  the  respiratory  center,  and 
was  difficult  to  count;  the  conjunctival  reflex  was  good;  the  dog  was 
unable  to  move  its  legs;  tremors  were  observed  in  the  leg  muscles,  the 
abdominal  muscles,  and  the  lips;  the  jaws  moved  incessantly,  as  if  the 
animal  was  gasping  for  breath;  the  dog  was  placed  in  a  warm  room  on 
a  piece  of  cotton.  The  animal's  condition  remained  unchanged  during 
the  remainder  of  the  day;  its  respiration  was  always  shallow  and 
irregular.  At  7  p.  m.  it  was  found  dead.  A  post-mortem  examination 
showed  the  following:  heart  distended  and  all  the  chambers  filled  with 
ante-mortem  clots;  these  clots  also  appeared  in  the  larger  blood  vessels; 
the  stomach  was  very  distended  and  was  filled  with  gas  and  undigested 
food ;  the  duodenum  was  filled  with  a  sticky,  bloody  mucus;  the  jejunum 
contained  a  dark  brown  mucous  substance;  the  blood  was  a  trifle 
darker  than  normal. 

In  this  case  asthenia  appeared  to  be  the  principal  symptom.  The 
action  of  the  drug  was  rapid,  but  did  not  cause  the  type  of  convulsions 
described  by  Filehne  as  following  a  rapid  action  of  the  drug. 

DOG  X  (female) 

Weight  of  dog,  ?  ^medium-sized) . 

December  7,  1916 —  Dog  fumigated  at  20°  C.  for  a  period  of  seven  hours 

and  fifty  minutes. 

Time  when  fumigation  was  begun,  2.10  p.  m. 

Time  when  fumigation  was  finished,  10  p.  m. 

'  Obs(M-vations:  ^i'h(^  animal  remaiiuid  quiet  during  the  fumigation,  and 
when  n^moved  from  the  tank  at  10  p.  m.  it  appeared  entirely  normal. 
No  symptoms  of  poisoning  appeared  until  two  days  later. 


440  Wallace  Larkin  Chandler 

December  9  —  At  9  a.  m.  the  animal  was  apparently  normal.  At  3  p.  m. 
it  exhibited  a  weakening  of  the  hind  legs,  and  walked  with  a  staggering, 
sprawling  gait,  showing  a  lack  of  coordination  of  the  muscles  of  the 
hind  legs;  it  had  recently  vomited.  At  5  p.  m.  the  animal  was  no  longer 
able  to  walk,  and  the  extensors  of  the  fore  legs  were  in  tetany.  At 
6  p.  m.  the  animal  was  no  longer  able  to  stand;  nystagmus  had  appeared, 
and  both  pupils  were  dilated,  the  left  more  widely  than  the  right;  the 
dog  drank  a  little  milk. 

December  10  —  The  general  condition  of  the  animal  was  about  the  same 
as  on  the  preceding  night.  Nystagmus  was  slight.  The  dog  drank 
milk  and  water  in  the  morning,  but  refused  both  food  and  drink  later 
in  the  day.  The  legs  were  extended;  there  was  nothing  definite  about 
the  extension  of  the  legs,  one  or  both  of  the  hind  legs  sometimes  being 
extended  and  rigid,  and  the  fore  legs  sometimes  being  thus  affected; 
at  times  the  tetany  would  last  for  a  long  period,  and  again  it  would 
be  of  short  duration.  At  times  the  head  was  drawn  strongly  backward, 
with  the  muscles  of  the  neck  rigid.  The  pupils  reacted  sHghtly  to 
light.     There  was  an  odor  of  nitrobenzene  on  the  animal's  breath. 

December  11  —  At  8  a.  m.  the  general  condition  of  the  animal  was  worse, 
but  it  was  still  conscious;  all  the  legs  were  rigidly  extended  for  minutes 
at  a  time,  and  the  head  was  drawn  backward;  when  this  condition  passed 
off  the  animal  was  left  prostrated ;  the  pupils  were  contracted  unequally. 
At  about  1  p.  m.  the  animal  passed  about  100  mils  of  dark  urine  (the 
first  passed  since  the  fumigation).  At  10  p.  m.  the  pupils  were  about 
normal;  the  animal  swallowed  a  very  little  milk  and  water  when  these 
were  placed  in  its  mouth;  when  disturbed,  the  animal  would  attempt 
to  use  its  legs,  and  this  resulted  in  a  tetanic  convulsion  involving  the 
muscles  of  the  legs  and  the  neck. 

December  12  —  During  the  night  the  animal  passed  about  100  mils  of 
very  dark  urine  (almost  like  black  coffee).  The  animal  had  regained  the 
ability  to  move  its  legs  a  httle,  tho  an  attempt  to  do  so  usually  threw 
them  into  tetany  of  the  type  described  above.  At  11  a.  m.  the  animal 
was  found  with  all  four  of  its  legs  in  constant  motion;  these  movements 
were  fairly  well  coordinated  and  rapid,  as  in  the  act  of  running  or  swim- 
ming; they  would  increase  in  rapidity  and  violence  until  the  animal  was 
thrown  into  a  convulsion  which  apparently  involved  every  muscle  of  the 


Physiological  Action  of  Nitrobenzene  Vapor  o.\  Animals     441 

body ;  the  legs  were  straight  and  the  head  was  drawn  down  under  the  body 
between  the  hind  legs ;  these  spasms  lasted  for  a  few  seconds,  during  which 
respiration  ceased  entirely;  as  the  spasms  passed  off,  the  animal  would 
give  a  short,  hollow  ciy  and  resume  the  running  movements,  tho  with 
evidence  of  exhaustion;  both  the  heart  rate  and  the  respiration  were 
rapid;  if  the  animal  was  lifted  up  or  its  legs  were  held  for  a  moment, 
the  swimming  or  running  movements  would  cease  for  a  time;  the  excite- 
ment was  very  similar  to  postanesthetic  excitement,  except  for  the  convul- 
sive periods.  At  3.30  p.  m.  the  animal  was  found  quiet  and  relaxed;  the 
respiration  was  35.  At  5  p.  m.  the  animal  swallowed  a  little  milk  and 
water  which  were  placed  in  its  mouth.  At  10  p.  m.  convulsions  were 
observed  which  were  of  tho  opisthotonos  type  except  that  the  muscles  of 
the  hind  legs  were  relaxed;  these  convulsions  appeared  at  intervals,  and 
were  induced  if  the  animal  was  disturbed. 

December  13  —  The  animal  was  able  to  swallow  but  a  very  little  liquid 
food  when  this  was  placed  in  its  mouth,  and  so  had  had  little  or  no 
nourishment.  It  lay  on  its  left  side,  with  its  head  diawn  back  on  its 
body,  its  fore  legs  extended  and  ligid,  and  one  or  both  of  its  hind  legs 
drawn  up  to  its  body.  The  rcspiiation  was  slow,  16-20,  and  was  deeper 
than  normal;  the  heart  was  irregular,  the  rate  being  aliout  160;  the 
tongue  and  the  conjunctiva  were  slightly  cyanotic.  Toward  evening 
the  heart  seemed  weaker;  the  animal  swallowed  a  little  milk;  it  had 
passed  very  littk^  in-ine,  and  the  little  that  was  passed  was  of  a  dark 
color.  The  cause  of  the;  color  of  this  urine  has  not  been  determined; 
it  was  not  hematoporphyrin,  l)y  the  spectroscopic  test. 

December  I4.  —  The  animal  was  found  dead.  It  died  sometime  after  11 
p.  m.  of  the  preceding  day.  A  post-mortem  examination  showed  the 
following:  lungs,  hypostatic  congestion  of  the  left  lobe;  liver,  congested 
and  dark  brown  in  color;  esophagus  and  stomach,  containing  a  little  clear 
mucous  substance:  all  other  organs  normal. 

DOG  XI  (female) 

Weight  of  dog,  ?  (smull,  about  8  kilograms). 

./(inimry  o,  W17  —  Dog  fumigated  at  20°  C  foi'  a  pcjriod  of  three  hours. 
'I'inic  when  funiigali(ji!  was  begun,  2.2o  p.  ni. 
Time  when  fumigation  was  finished,  5.25  p.  ni. 


442  Wallace  Larkin  Chandler 

Observations:  The  animal  remained  fairly  quiet  duririg  the  fumigation 
and  was  removed  from  the  tank  at  5.25  p.  m.  in  an  apparently  normal 
condition.  It  was  lively,  ate  heartily,  and  showed  no  symptoms  of  poison- 
ing.    No  symptoms  developed  during  the  next  three  days. 

January  8 — ^  When  seen  at  11  a.  m.  the  animal  was  apparently  well  and 
normal.  At  5  p.  m.,  however,  it  exhibited  a  loss  of  coordination  of  the 
muscles  of  the  hind  legs,  and  had  vomited  at  some  time  previously.  At 
8  p.  m.  it  was  unable  to  stand.  , 

January  9  —  The  animal  was  found  lying  on  its  left  side  in  a  helpless 
condition;  when  placed  on  its  right  side,  it  struggled  violently  until  it 
regained  the  left  side;  it  refused  cooked  meat  and  water,  but  drank  a 
little  milk.  At  12  m.  nystagmus  was  observed;  the  pupils  were  normal; 
the  animal  ate  a  Httle  cooked  liver.  By  3  p.  m.  nystagmus  was  very 
marked;  the  pupils  were  normal;  knee-jerk  reflex  was  good  in  both  legs. 
When  seen  at  7  p.  m.  the  animal  tried  to  stand  on  its  feet,  but  its  hind 
legs  were  apparently  paralyzed;  when  it  did  not  succeed,  it  at  once 
began  to  howl. 

January  10  —  The  condition  of  the  animal  was  about  the  same.  It  lay 
continually  on  its  left  side  and  was  unable  to  move  its  body.  There 
was  a  certain  amount  of  rigidity  of  the  leg  muscles  at  times,  but  this 
was  not  well  marked.  Knee-jerk  reflex  was  good.  The  animal  ate 
cooked  meat,  and  drank  milk  but  no  water.  Nystagmus  was  not  notice- 
able. 

January  11 — The  condition  of  the  animal  was  slightly  improved.  It 
could  move  its  legs  a  little.  It  ate  meat  and  drank  milk,  but  refused 
water  (it  was  given  meat  and  milk  twice).     The  pupils  were  normal. 

January  12  —  The  condition  of  the  animal  was  much  improved.  It  was 
able  to  lift  its  body  on  its  fore  legs  and  crawl  about  the  cage,  but  the 
fore  legs  weakened  quickly.  The  animal  passed  urine  which  was  very 
dark.     It  ate  meat  greedily  and  drank  some  water. 

January  13  —  The  condition  was  much  improved.  The  animal  was  able 
to  walk  about  very  well,  but  its  legs  seemed  weak  and  gave  way  at 
times. 

January  I4  — ■  The  condition  was  still  further  improved. 


Physiological  Actiox  of  Nitrobenzene  Vapor  on  Animals    443 

January  15  —  The  condition  was  apparently  normal.  The  dog  was 
turned  loose  with  the  other  dogs. 

In  this  case  no  definite  convulsions  were  observed,  the  dominant 
symptom  being  paralysis  such  as  was  reported  by  Filehne  as  following 
a  slow  action  of  the  drug. 

dog  xviii  (female) 
Weight  of  dog,  ?  (small,  rather  thin;  heavy  Demodex  infection). 

April  23,  1917 —  Bog  fumigated  at  23°  C.  for  a  period  of  four  hours. 
Time  when  fumigation  was  begun,  2  p.  m. 
Time  when  fumigated  was  finished,  6  p.  m. 

Observations:  The  animal  was  I'cstless  for  a  time  after  being  placed 
in  the  fumigation  chamber,  but  soon  became  quiet.  It  was  removed 
apparently  unharmed,  and  never  developed  any  symptoms  of  poisoning 
as  the  result  of  this  fumigation. 

Maij  16,  1917  —  Dog  fumigated  a  second  time,  this  time  at  20°  C.  for  a 
period  of  five  and  one-half  hours.  (The  dog  was  slightly  fatter  than 
when  first  fumigated.) 

Time  when  fumigation  was  begun,  1.10  p.  m. 
Time  when  fumigation  was  finished,  6.40  p.  m. 

Observations:  As  before,  the  animal  was  a  bit  restless  when  first 
placed  in  the  tank,  but  it  soon  became  accustomed  to  its  new  environment 
and  became  quiet.  It  was  removed  from  the  tank  apparently  unharmed, 
showing  no  signs  of  nitrobenzene  poisoning  and  drinking  water  freely. 

May  17  —  No  symptoms  had  developed. 

May  18  —  The  animal  was  found  with  its  hind  legs  paralyzed,  and  there 
were  evidences  of  its  having  thrashed  about  during  the  night.  It 
drank  milk  and  water  freely  and  ate  meat.  It  was  placed  in  a  padded 
cage. 

May  19  —  The  general  condition  of  the  animal  was  worse.  It  could 
rai.se  its  head  and  wag  its  tail,  but  its  legs  were  useless.  It  ate  and  drank. 
This  condition  remained  al)Out  the  same  until  May  22,  when  some  improve- 
ment was  noticed. 


444  Wallace  Larkin  Chandler 

May  23  —  The  animal  was  found  with  its  body  raised  on  its  fore  legs, 
swaying  from  side  to  side,  apparently  making  efforts  to  stand  up  but 
its  hind  legs  were  useless.  The  animal  had  not  defecated  since  being 
placed  in  the  cage,  altho  it  was  taken  out  several  times  for  this  purpose. 

May  21  —  The  animal  was  very  much  improved..  It  could  use  its  fore 
legs  very  well  and  had  some  use  of  its  hind  legs,  but  when  it  attempted 
to  walk  it  staggered  and  fell,  or  rather  tumbled  to  the  floor,  striking 
its  jaws  against  the  floor  with  considerable  force.  It  appeared  very 
nervous,  and  was  always  moving  and  fidgeting  about,  apparently 
unable  to  remain  quiet  at  all.  The  animal  defecated  for  the  first  time 
since  the  beginning  of  the  experiment. 

May  26  —  The  general  condition  of  the  animal  was  about  the  same.  It 
was  taken  out  on  the  lawn  for  exercise.  In  standing,  its  hind  legs  were 
spread  far  apart.  It  was  unable  to  walk  or  to  run,  but  it  actually 
tumbled  along,  jumping  high  into  the  air  and  coming  down  on  its  head 
or  its  back,  turning  somersaults,  or  tumbling  over  sidewise.  This  dog 
was  by  nature  playful  and  it  had  lost  none  of  its  playfulness  as  the  result 
of  the  fumigation;  its  efforts  to  play  always  resulted  in  its  throwing 
itself  violently  about.  An  interesting  observation  was  the  attempt  of 
the  dog  to  go  toward  any  one  when  called;  it  made  better  progress  in 
attempting  to  go  in  the  opposite  direction.  It  apparently  was  con- 
fused as  to  distances,  and  was  wholly  unable  to  make  progress  in  a 
straight  line.^" 

RABBITS  I  AND  II,  AND  GUINEA  PIGS  I  AND  II 

November  2,  1916  —  Animals  fumigated  at  22°  C.  for  a  period  of  nine 

hours. 

Time  when  fumigation  was  begun,  10.30  a.  m. 

Time  when  fumigation  was  finished,  7.30  p.  m. 

Observations:     These  animals  were  apparently  normal  when  removed 
from  the  tank,  and  never  developed  any  symptoms  afterward. 

'"This  animal  was  killed  in  February,  1918,  and  a  histological  examination  of  the  cerebellum  revealed 
a  striking  absence  of  Purkinje  cells.  Only  from  5  to  10  per  cent  of  the  number  found  in  a  normal  dog 
were  present.  The  cont.-^ur  of  t'-e  cerebellum  was  aoparently  normal,  and  those  Purkin.ie  cells  which 
were  present  were  scaifere!  f'\lr! ,-  uniformly  thruoit  t^ie  cerebellum.  The  condition  of  the  animal  had 
never  improved  very  markedly;  and  while  it  had  learned  many  riew  tricks  regarding  locomotion,  its 
actions  were  always  typically  those  of  a  cerebellar  animal. 


Physiolocical  Action  of  Nitrobenzene  Vapok  ox  Animals    445 

November  IS,   1910  —  Animals   fumigated   a  second   time,   tiiis   time  at 
24°  C.  for  a  period  of  nine  hours  and  thirty  minutes. 
Time  when  fumigation  was  begun,  10  a.  m. 
Time  when  fumigation  was  finished,  7.30  p.  m. 

Observations:  As  before,  these  animals  showed  no  effects  of  the  drug, 
either  during  the  fumigation  or  afterward. 

rabbit  III  AND  GUINEA  PIG  III 

Dec.  22,  1916  —  These  animals  were  placed  together  in  the  fumigation 
chamber  at  5.15  p.  m.,  and  were  allowed  to  remain  there  until  4  p.  m. 
on  December  23.  The  temperature  of  the  chamber  remained  constant 
at  20°  C.  during  the  first  seven  hours,  then  it  gradually  dropped  until 
at  the  end  of  the  next  seven  hours  it  was  15°  C,  and  then  it  rapidly 
rose  to  20°  C.  again.  During  the  second  seven  hours  no  air  was  intro- 
duced into  the  tank. 

Observations:  The  animals  nestled  together  and  remained  quiet  dur- 
ing the  entire  experiment.  A  string  was  tied  to  the  rabbit's  leg,  and  every 
hour  or  so  the  anunal's  reflexes  were  tested.  They  remained  good.  Both 
animals  were  a  trifle  stupid  when  they  were  removed  from  the  tank. 
They  were  offered  food  and  water,  but  would  not  drink  and  barety  nibbled 
at  the  food.  Suddenly  the  guinea  pig  fell  on  its  right  side  and  was  unable  to 
regain  its  feet.  When  placed  on  its  left  side,  it  immediately  turned  again 
to  the  right  side.  Violent  tremors  were  observed  in  all  its  muscles,  and 
presently  it  was  seized  with  convulsions;  all  the  legs  were  rigid  and  the  head 
was  drawn  back  on  the  body.  This  spasm  lasted  but  a  few  seconds,  and 
when  it  ended  the  animal  shook  itself  violently  and  then  executed  running 
movements  similar  to  those  described  in  dogs.  These  movements  were 
extremely  rapid  and  lasted  until  another  convulsion  appeared.  At  8  p.  m. 
the  guinea  pig  seemed  to  be  recovering  and  was  able  to  raise  itself  on  its 
fore  legs.  It  remained  quiet  for  some  time,  and  when  observed  the  next 
moi-ning  it  was  dead.  A  post-mortem  examination  showed  the  lungs  to 
be  distended  and  the  air  passages  were  filled  with  blood;  the  blood. was 
dark   brown;  the  liver  was  congested;  the  other  organs  were  normal. 

At  the  end  of  two  hours  the  rabbit  had  developed  no  symptoms,  and  at 
about  G  p.  m.  it  was  again  placed  in  the  fumigation  (^hambei-.  The  animal 
reacted  to  the  jerk  of  the  string  until  about  9  p.  m.     It  was  then  removed 


446  Wallace  Larkin  Chandler 

from  the  tank  and  was  found  to  be  in  a  stupor;  it  was  wholly  relaxed  and 
perfectly  reactionless.  It  remained  in  this  condition  for  a  few  hours, 
and  then  died,  without  any  signs  of  convulsions. 

GRAY  RATS 

{Mus  decumanus) 

Five  young  rats  were  placed  together  in  a  large  wooden  box  having  a 
capacity  of  10  cubic  feet.  Fifteen  drops  of  nitrobenzene  were  placed  on 
a  strip  of  cheesecloth  and  the  cloth  was  suspended  in  the  box,  which  was 
then  closed  for  twelve  hours.  At  the  end  of  that  time  the  rats  were 
removed.  The  animals  were  all  perfectly  anesthetized,  and  three  of 
them  were  reactionless.  One  died  five  hours  later,  and  another  was 
killed  for  the  purpose  of  examining  its  blood;  both  of  these  had  dark 
brown  blood.  All  the  other  three  exhibited  either  right  or  left  rotatory 
(pinwheel)  movements;  one  of  them  was  seen  to  roll  over  and  over  for 
several  feet  before  becoming  exhausted.  Two  of  these  remaining  three 
died  without  showing  other  symptoms,  and  one  recovered  (at  least  tem- 
porarily) and  escaped. 

Two  adult  rats  of  the  same  species  were  fumigated  together  m  the 
regular  fumigation  chamber  for  three  and  one-half  hours  at  a  temperature 
of  23.5°  C.  When  removed  from  the  tank  they  were  apparently  unharmed. 
Both  of  these  died  two  days  later,  probably  from  lack  of  nourishment 
since  it  was  impossible  to  induce  them  to  eat  while  in  captivity. 

WHITE  RATS 

It  was  found  that  white  rats  could  not  stand  a  fumigation  at  ordinary 
temperatures  for  longer  than  from  one  and  one-half  to  two  hours.  How- 
ever, the  rats  used  in  these  experiments  were  infected  with  trypanosomes 
and 'spirochetes,  and  this  fact  may  have  had  something  to  do  with 
hastening  the  action  of  the  drug.  The  rats  that  were  still  alive  when 
removed  from  the  tank  showed  only  paralysis  and  usually  died  very 

quickly. 

CAT  X 

September  26,  1916  —  Cat  fumigated  at  17°  C.  for  a  period  of  five  hours. 
Time  when  fumigation  was  begun,  11.50  a.  m. 
Time  when  fumigation  was  finished,  4.50  p.  m. 


Physiological  Action  of  Nitrobenzenp:  Vapor  on  Animals    447 

Observations:  -The  animal  lay  down  and  remained  quiet  foi-  about 
three  hours,  and  then  became  restless  for  a  time  but  soon  became  quiet 
again.  When  first  removed  from  the  tank  the  animal  appeared  well  and 
started  to  walk  away,  but  lost  control  of  its  hind  legs  and  tumbled  about 
for  a  moment,  then  became  excited.  It  vomited  (chunks  of  meat  which 
had  not  started  to  digest),  and  then  lay  prostrate  for  about  one-half  hour. 
The  lips  and  the  tongue  were  cyanotic;  the  pupils  were  dilated  and  did 
not  react  to  hght.  The  animal's  condition  improved  shortly  and  it  again 
appeared  well.  On  the  following  morning  a  quantity  of  sticky,  l)loody 
fec(>s  was  found  in  the  animars  cage;  no  further  symptoms  of  poisoning 
had  developed,  however,  nor  did  any  symptoms  appeal-  during  the  next 
four  days.     At  the  end  of  this  tinu^  the  cat  was  found  dead. 

CAT  XVIII 

October  5,  1917  —  Cat  fumigated  at  22.5°  C.  for  a  period  of  three  hours. 
Time  when  fumigation  was  begun,  9  a.  m. 
Time  when  fumigation  was  finished,  12  m. 

Observations:  The  animal  remained  quiet  during  the  fumigation  and 
was  removed  from  the  tank  apparently  unharmed,  but  became  sUghtly 
stupid  toward  evening.  A  sample  of  blood  was  taken  and  examined. 
It  was  dark  brown  and  showed  methemoglobin  by  spectroscopic 
analysis. 

October  6  —  The  animal  was  found  with  well-advanced  symptoms  of  nitro- 
benzene poisoning  —  nystagmus,  paralysis  of  the  muscles  of  the  hind 
legs,  and  periods  of  excitation  followed  l)y  prostration.  The  cat  re-fused 
milk,  nor  was  it  possible  to  place  any  in  its  mouth.  A  sample  of  blood 
drawn  from  the  ear  was  coffee-colored  and  showed  the  same  sixictrum 
as  on  the  preceding  day. 

October  7  —  The  condition  of  the  animal  remained  about  the  same.  It 
lay  on  its  side  entirely  helpless  and  apparently  unconscious. 

October  8  —  TJie  (x>ndition  gradually  grew  worse  and  the  animal  died 
toward  evening.  A  post-mortem  examination  showed  a  pronounced 
congestion  of  lungs  a'nd  viscera,  and  the  blood  vessels  of  the  neck  were 
turgid  with  blood. 


448  Wallace  Larkin  Chandler 

HEN   V 

March  21,  1917  —  Hen  fumigated  at  30°  C.  for  a  period  of  six  and  one- 
half  hours. 

Time  when  fumigation  was  begun,  1  p.  m. 
Time  when  fumigation  was  finished,  7.30  p.  m. 

Observations :  The  bird  was  removed  from  the  tank  apparently  normal 
except  that  it  appeared  a  Uttle  stupid.  The  feces  were  formed  when  the 
bird  was  first  placed  in  the  fumigation  chamber,  but  those  deposited 
immediately  after  the  bird  was  removed  from  the  tank  were  soft,  stringy, 
and  slightly  bloody. 

March  22  —  The  bird  appeared  normal  except  that  the  neck  feathers 
were  constantly  ruffled  —  a  condition  which  induced  such  a  belligerent 
attitude  on  the  part  of  the  other  birds  in  the  cage  that  this  hen  had  to 
be  placed  by  itself.  No  further  symptoms  developed  until  March  27, 
on  which  day  the  bird  was  found  lying  prone  on  its  side  and  apparently 
unable  to  stand.  When  the  bird  was  lifted  it  exhibited  a  circular  rotation 
of  head  and  neck;  and  when  it  was  placed  on  its  feet,  the  legs  stiffened, 
throwing  the  bird  backward. 

March  28  —  The  bird's  general  condition  was  about  the  same.  Nothing 
new  was  especially  noted. 

March  29  —  The  bird's  condition  had  grown  worse.  The  skin  was  cold, 
and  the  bird  was  placed  in  a  warm  room.  There  appeared  frequent 
periods  of  excitation,  during  which  the  bird  thrashed  about  a  good  deal. 

March  30  —  The  bird  was  found  lying  on  its  left  side,  with  its  left  leg  ex- 
tended and  rigid  and  its  right  leg  exhibiting  violent  tremors.  The  respiration 
was  normal.  The  head  was  moving  by  jerks  sidewise.  When  placed  on 
its  feet  the  bu-d  lunged  forward  instead  of  backward.  At  2  p.  m.  the 
bird  swallowed  some  water  when  its  head  was  placed  in  water  and  then 
released;  it  also  swallowed  a  little  moistened  bread  when  this  was  placed 
in  its  mouth.  Each  attempt  at  swallowing  was  followed  by  excitation, 
during  which  the  head  was  drawn  back  rigidly  between  the  wings  or 
revolved  slowly,  describing  broad  circles.  Wlien  placed  on  its  feet  the 
bird  lunged  backward. 

March  31  —  The  l^ird  was  again  fed  as  described. 


Physiological  Action  of  Nitrobenzene  Vapor  on  Animals    449 

April  1  —  The  eondition  was  about  the  same,  altho  the  bird  appeared 
stronger.  When  placed  on  its  feet  it  attempted  to  walk,  but  the  legs 
stiffened  and  the  bird  was  thrown  foi'wai'd. 

This  bird  never  fully  recovered.  It  was  able  to  squat  on  its  feet  after 
a  time,  but  refused  to  walk;  wh(>n  urged,  it  took  two  or  three  rapid  steps 
and  then  tumbled  over  forward.  It  was  unable  to  remain  on  a  perch, 
faUing  either  forward  or  backwai'd.  The  wing  movements  were  well 
coordinated.  Foi'  a  long  time  the  bird  was  unable  to  eat  without  assist- 
ance, but  it  was  finally  taught  to  do  so.  The  bird  was  killed  on  June  11, 
in  order  to  make  an  examination  of  \\\v  biain  tissues. 

HEN   VI 

March  ,12,  1917  —  Hen  fumigated  at  27-28°  C.  for  a  pei'iod  of  eleven  hours. 
Time  when  fumigation  was  begun,  10  a.  m. 
Time  when  fumigation  was  finished,  9  p.  m. 

Ob.servations:  The  bird  was  removed  fiom  the  tank  ajiparently 
unharmed.     The  feces  were  as  described  for  Hen  V. 

March  '23  —  The  bird  was  apparently  well,  but  not  very  active.  It  ate 
cracked  corn  and  di"ank  water.     The  neck  feathers  were  ruffled. 

March  24  —  Wlien  first  seen  on  this  day  (at  8  a.  m.)  th(>  bird  appeared 
normal.  It  was  taken  from  the  cage  and  placed  on  the  floor.  It  was 
able  to  walk  and  run  very  well,  but  suddenly  showed  a  tendency  to  give 
way  to  the  left  side;  it  flew  to  a  perch  18  inches  high,  but  was  unable 
to  retain  its  position;  it  fatigued  very  easily.  At  10  a.  m.  the  attendant 
reported  that  the  bird  had  been  in  convulsions;  it  appeared  normal 
except  that  it  was  a  little  dull.  At  3  p.  m.  the  bird  appeared  to  be  drowsy; 
it  was  removed  from  the  cage  and  placed  on  its  feet;  it  stood  swaying 
from  side  to  side;  when  it  attempted  to  walk,  it  staggered  and  then  swayed, 
backward,  taking  several  steps  in  an  effort  to  catch  itself;  after  this  excite- 
ment its  head  was  bent  back  between  its  wings.  At  4  p.  m.  the  bird  was 
found  lying  on  its  side  and  was  unable  to  stand;  when  it  was  [)icked  up, 
its  head  rotated  in  a  ciicle,  sweeping  the  back  and  the  wings.  At  4.15 
about  an  ounce  of  clear  fluid  came  from  the  bird's  mouth.  At  ")  p.  m. 
periods  of  excitation  were  observed;  the  legs  wei'e  in  violent,  motion  as 
in  the  act  c)f  nuining,  and  ihe  head  shook  viok'iitly  as  if  the  bird  were 


450  Wallace  Larkin  Chandler 

trying  to  dislodge  something  in  it;  these  periods  of  excitation  were  of 
short  duration  and  were  followed  by  periods  of  prostration  lasting  for 
several  minutes,  during  which  the  muscles  were  wholly  relaxed  and  the 
neck  was  limp;  then  with  a  sudden  jerk,  the  period  of  excitation  would 
appear  again;  this  excitation  could  always  be  elicited  by  disturbing  the 
bird;  when  the  bird  was  placed  on  its  feet,  the  legs  stiffened  and  threw 
the  body  backward;  the  legs  were  bluish  (they  were  pink  normally); 
frequent  movements  of  the  gullet  were  observed,  these  movements  often 
involving  the  mouth.  At  5.15  another  ounce  of  clear  fluid  came  from 
the  mouth.  At  6  p.  m.  the  bird  made  a  violent  effort  to  regain  its  feet; 
the  wing  movements  were  well  coordinated  but  weak;  this  effort  was 
followed  by  excitation  during  which  the  legs  moved  violently  as  described 
above. 

March  25  —  At  9  a.  m.  the  bird's  condition  was  worse;  there  was  constant 
rotation  of  head  and  neck;  convulsions  occurred  as  before;  when  placed 
on  its  feet,  the  bird  squatted  quietly  for  a  moment  and  discharged  a  large 
quantity  of  watery  fluid  from  the  anus,  and  then  went  into  convulsions 
again.  At  5  p.  m.  the  bird  was  apparently  better,  and  was  resting  quietly 
in  an  upright  position.  At  7  p.  m.  the  bird  was  found  in  convulsions; 
the  head  was  bent  firmly  on  the  venter  (almost  between  the  legs);  the 
bird  had  no  control  of  any  muscle. 

March  26  —  In  the  morning  the  bird  was  found  with  all  muscles  relaxed 
except  the  neck  muscles,  which  were  rigid  and  held  the  head  firmly  against 
the  venter.  At  6  p.  m.  the  bird  was  found  dead.  A  post-mortem  exami- 
nation revealed  a  strong  odor  of  nitrobenzene  in  all  the  organs;  the  crop 
and  the  gizzard  were  filled  with  cracked  corn,  which  had  not  started  to 
digest  altho  the  bird  had  had  nothing  to  eat  during  the  preceding  sixty  hours : 
the  other  organs  were  about  normal. 

HEN   VII 

March  25,  1917  — Ren  fumigated  at  25°  C.  for  a  period  of  eight  hours. 
Time  when  fumigation  was  begun,  2.15  p.  m. 
Time  when  fumigation  was  finished,  10.15  p.  m. 

Observations:  When  removed  from  the  tank  the  bird  was  a  trifle 
stupid  but  was  otherwise  normal.  It  developed  no  symptoms  of  poisoning 
until  March  30. 


Physiological  Action  of  Nitrobenzene  Vapor  on  Animals    451 

March  31  —  The'  bird  showed  symptoms  very  similar  to  those  described 
for  Hen  VI. 

April  1  —  The  bird  was  found  dead. 

On  March  27  (two  days  after  the  fumigation)  this  hen  laid  an  egg- 
The  egg  was  opened  on  March  29 ;  a  strong  odor  and  a  very  characteristic 
taste  of  nitrobenzene  were  detected  in  the  yolk,  but  the  white  did  not 
contain  more  than  a  trace  of  the  chemical.  This  phenomenon  can  easily 
be  explained  by  the  fact  that  nitrobenzene  is  soluble  in  fats,  but  is  scarcely 
soluble  at  all  in  the  white  of  eggs. 

HEN    VIII    AND    ROOSTER    III 

May  10,  /5/ 7  — Birds  fumigated  together,  at  23°  C,  for  a  period  of 

eight  hours. 

Time  when  fumigation  was  begun,  2  p.  m. 

Time  when  fumigation  was  finished,  10  p.  m. 

Observations:  The  rooster  was  found  dead  at  the  end  of  the 
fumigation  period.  The  hen  appeared  slightly  stupid  when  removed 
from  the  tank  and  became  easil}^  fatigued,  but  was  otherwise  normal. 
No  further  symptoms  developed  in  the  hen  until  on  May  14,  when  it 
was  seen  to  stagger  on  attempting  to  run.  During  the  next  few  da3^s 
the  bird  was  very  stupid.  It  did  not  eat  much,  staggered  in  attempting 
to  walk,  and  fatigued  easily. 

May  25  —  The  condition  of  the  bird  was  apparently  normal  again.  No 
further  symptoms  ever  developed  in  this  bird. 

PIGEONS   IV   AND    V 

June  11,  1917  —  Birds  fumigated  at  24°  C.  for  a  period  of  six  hours. 
Time  when  fumigation  was  begun,  1.10  p.  m. 
Time  when  fumigation  was  finished,  7.10  p.  m. 

Observations:  The  l)irds  wen>  apparently  normal  when  removed  from 
the  tank.     They  could  fly  and  run  easily. 

June  12  —  One  of  the  birds  showed  the  following  symptoms:  it  was 
unable  to  fly,  tho  the  wing  movements  were  fairly  well  coordinated; 
in  attempting  to  walk,  it  lunged  forward  and  tumbled  on  its  head;  there 
was  rotation  of  head  and  neck. 


452  Wallace  Larkin  Chandler 

June  13  —  The  bird  showing  on  June  12  the  symptoms  described,  flew 
the  length  of  the  room  along  the  floor,  its  head  touching  the  floor.  It 
probably  had  the  use  of  its  wings  but  could  not  direct  the  flying  move- 
ments. The  other  bird  showed  symptoms  of  poisoning  on  this  day. 
Both  the  birds  were  killed  about  noon,  and  the  nervous  tissues  were 
fixed  as  described  earlier  (page  429). 

OBSERVATIONS  OF  THE  ACTION  OF  NITROBENZENE  ON  INSECTS 

During  the  fumigation  of  the  animals  a  number  of  external  parasites 
dropped  from  the  hosts.  These  were  collected  at  the  end  of  the  fumigation 
period  and  observations  were  recorded  regarding  the  action  of  the  drug 
on  them.     Some  of  these  observations  were  as  follows: 

fleas 

Eighty-three  fleas  of  the  genus  Ctenocephalus  were  recovered  from 
the  bottom  of  the  tank  after  the  fumigation  of  Cat  X  (fumigated  for 
five  hours  at  17°  C).  Most  of  these  began  to  show  signs  of  life  in  about 
one  and  one-half  hours;  they  were  put  into  a  glass  tube  and  placed  in 
a  warm  room,  and  in  about  twelve  hours  all  the  fleas  had  recovered  with 
the  exception  of  about  one-half  dozen,  which  were  stuck  fast  to  the  tube. 

Nineteen  fleas  of  the  same  genus  were  recovered  in  a  stupefied  con- 
dition from  the  bottom  of  the  tank  after  the  fumigation  of  Dog  I  (second 
fumigation,  six  hours  at  23°  C).  Three  of  these  .showed  signs  of  life, 
and  most  of  them  recovered  during  the  next  twelve  hours. 

Seventy-six  fleas  of  the  same  genus  were  recovered  from  the  tank  after 
the  fumigation  of  three  kittens  for  a  period  of  four  hours  at  22°  C.  Some 
of  these  fleas  showed  signs  of  life  when  removed  from  the  tank,  and  twenty 
of  them  recovered  and  lived  for  several  days. 

BITING  lice 

A  large  number  of  biting  lice  (Trichodectes  suhrostratus)  were  recovered 
in  a  stupefied  condition  from  the  bottom  of  the  tank  after  the  fumigation 
of  Cat  X  (fumigated  for  five  hours  at  17°  C.) .  Nearly  all  of  these  recovered 
during  the  next  twelve  hours. 

A  large  number  of  the  biting  lice  of  poultry  {Menopon  gallinae,  Menopon 
stramineum,  Lipeurus  heterographus,  and  Goniocotes  gigas)  were  recovered 


Physiological  Action  of  Nitrobenzene  Vapor  on  Animals    453 

from  the  tank  after  the  fumigation  of  Hen  V  (fumigated  for  six  and  one- 
half  hours  at  30°  C).  The  hce  were  all  apparently  dead,  showing  no 
signs  of  life,  at  the  end  of  twelve  hours.  At  the  end  of  eighteen  hours, 
however,  some  of  them  were  seen  to  be  moving  their  legs.  In  Goniocotes 
these  movements  were  lapid,  and  were  very  similar  in  character  to  the 
movements  of  the  legs  of  poisoned  guinea  pigs. 

Approximately  three  hundred  biting  lice,  representing  six  species, 
were  recovered  from  th(^  tank  after  the  fumigation  of  a  chicken  for  a 
period  of  one  and  one-half  hours  at  20°  C.  Some  of  these  insects  showed 
signs  of  life  when  removed  from  the  tank,  and  nearly  all  of  them  recovered 
entirely  during  the  next  few  hours. 

Nineteen  specimens  of  biting  lice  were  recovered  from  the  bottom  of 
the  tank  after  the  fumigation  of  Hen  VI  (fumigated  for  eleven  hours  at 
27-28°  C).  None  of  these  showed  signs  of  Hfe  when  taken  from  the  tank, 
and  none  recovered. 

THE    FOLLICULAR   MITE 

(Demodex  folUculorum) 

Two  or  three  cases  of  mild  infection  of  demodecic  scabies  in  dogs 
apparently  cleared  up  after  fumigations  for  long  periods  at  low  tem- 
peratures. One  of  these  cases  was  Dog  V,  which  was  fumigated  twice  — 
once  for  twelve  hours  at  20°  C,  and  once  for  five  hours  at  20°  C.  These 
observations  led  to  the  following  experiments  for  the  purpose  of  determining 
the  value  of  nitrobenzene  in  controlling  demodecic  scabies. 

A  special  fumigation  chamber  was  constructed  in  such  a  way  that 
the  animal's  nose  passed  thru  a  rubber  collar  and  remained  on  the  outside 
of  the  chamber,  and  an  attempt  was  made  to  fumigate  the  body  of  the 
animal  without  permitting  it  to  inhale  a  large  amount  of  the  vapor. 
Morphine  and  chloral  hydrate  were  given  in  order  to  cause  the  animal 
to  remain  quiet  during  the  fumigation.  A  six-hour  fumigation  under 
these  special  conditions  had  no  effect  on  the  mites,  nor  was  it  possible 
to  induce  dogs  to  remain  quiet  without  giving  them  large  doses  of  the 
narcotics.     The  method  was  therefore  abandoned. 

Small  pieces  of  skin  heavily  infected  with  D.  folUculorum  var.  cards 
were  placed  in  a  petri  dish  in  which  there  was  a  drop  or  so  of  nitrobenzene. 
The  dish  was  then  kept  at  a  temperature  of  30°  C.  for  six  hours.  At  the 
end  of  that  time  the  mites  weie  still  alive. 


454  Wallace  Larkin  Chandler 

It  was  observed  that  a  33-per-cent  solution  of  nitrobenzene  in  olive 
oil  would  have  no  apparent  effect  on  dogs  if  applied  externally  and  if 
the  animal  was  allowed  to  remain  in  the  open  air,  in  spite  of  the  fact 
that  an  external  application  of  a  solution  of  this  strength  invariably 
killed  cats  and  rabbits.  Hence  a  small,  short-haired  dog  ^^  having  a 
heavy  infection  of  Dernodex  folliculorum  was  bathed  thoroly  and  fre- 
quently with  the  33-per-cent  solution ;  but  no  improvement  in  the  condition 
of  the  animal  could  be  noticed. 

OBSERVATIONS  OF  THE  ACTION  OF  NITROBENZENE  ON  INTERNAL 

PARASITES 

GAPEWORMS 

{Syngamus  trachealis) 

Three  chicks  showing  symptoms  of  gapes  were  fumigated  together 
for  two  hours  at  25-26°  C.  on  June  23,  1917.  Two  of  the  chicks  developed 
symptoms  of  nitrobenzene  poisoning  on  June  24.  One  of  these  died  on 
June  25  and  the  other  on  June  26.  Two  nearly  mature  pairs  of  Syngamus 
trachealis  were  recovered  alive  from  the  trachea  of  the  former,  and  three 
living  pairs  were  taken  from  the  trachea  of  the  latter.  The  third  chick 
developed  typical  symptoms  of  nitrobenzene  poisoning  on  June  26.  By 
July  8,  however,  this  chick  had  fully  recovered  from  the  effects  of  the 
drug,  and  it  had  also  recovered  from  the  gapes  by  July  8.  The  recovery 
from  the  gape  worms,  however,  cannot  be  attributed  to  the  "action  of  the 
drug  on  the  worms,  since  with  its  improved  environment  it  would  in  all 
probability  have  recovered  anyway, 

INTESTINAL   WORMS 

A  number  of  roundworms  {Belascaris  marginata)  were  found  in  the 
feces  of  Dog  III  on  the^  morning  following  a  fumigation  for  five  hours 
at  18°  C.  These  worms  were  obsei^ved  to  be  dead  and  their  death  was 
attributed  to  the  action  of  the  drug.  However,  roundworms  of  the  same 
species  were  recovered,  very  much  alive,  from  the  intestines  of  nearly  all 
the  dogs  examined,  even  in  cases  following  long  periods  of  fumigation. 

"  The  solution  was  not  tried  on  long-haired  dogs,  nor  was  a  stronger  solution  tried.     The  animal  did 
HOC  lick  the  solution  off,  presumably  because  of  the  burning  taste  of  nitrobenzene. 


Physiological  Action  ok  Nitrobenzene  Vapor  on  Animals    455 

coccidia 

Specimens  of  Eimeria  avium  in  the  oocyst  stage  were  recovered  from 
the  feces  of  Hen  VII.  These  oocysts  appeared  normal,  and  the  develop- 
ment of  sporocysts  and  sporozoites  occurred  as  usual. 

EXPERIMENT  TO  DETERMINE  THE  ACTION  OF  NITROBENZENE  ON 
DIGESTIVE  FUNCTIONS 

Post-mortem  examinations  of  animals  poisoned  by  nitrobenzene  usually 
revealed  the  fact  that  food  which  had  been  in  the  stomach  for  some  days 
had  not  started  to  digest.  A  special  experiment  was  therefore  conducted 
in  order  to  check  these  observations. 

At  11  a.  m.  on  October  3,  1916,  four  kittens,  all  from  the  same  litter, 
were  each  given  an  equally  large  portion  of  boiled  hamburg  steak.  At 
11.10  three  of  these  kittens  were  placed  in  the  fumigation  chamber,  where 
they  were  fumigated  for  four  hours  at  a  temperature  of  22°  C. 

At  2.20  p.  m.  (three  hours  and  ten  minutes  after  the  fumigation  was 
begun)  one  of  the  animals  died.  The  other  two  were  removed  from  the 
tank  at  3.10  and  showed  well-advanced  symptoms  of  poisoning.  One 
of  them  died  at  about  4  p.  m.  and  the  other  at  about  5  p.  m. 

At  about  5.30  p.  m.  the  control  kitten  was  killed  with  chloroform  and 
a  post-mortem  examination  was  made  of  all  four  animals.  The  stomachs 
of  the  animals  that  had  been  fumigated  contained  the  full  amount  of 
the  hamburg  steak  eaten,  and  in  no  case  had  it  even  started  to  digest. 
The  digestion  of  the  hamburg  steak  in  the  stomach  of  the  control  animal 
was  well  advanced. 

EXPERIMENTS  TO  DETERMINE  THE  ACTION  OF  NITROBENZENE  ON  BLOOD 

BLOOD    COUNTS 

Two  pups,  from  the  same  litter  and  about  three  months  old,  were 
kept  under  similar  conditions,^^  ^ji^j  blood  counts  were  made  on  each  for 
several  days  in  order  to  determine  the  normal  counts.  Each  animal  was 
then  fumigated,  on  different  days  and  for  different  periods  of  time,  and  the 
blood  counts  were  continued.  Always,  of  course,  as  soon  as  an  animal 
developed  pronounced  symptoms  of  poisoning  it  refused  food,  and  this 
interfered  with  exact  compaiisons.  The  lesults  of  these  experiments 
are  indicated  in  the  following  tables: 

'2 These  animals  were  fwi  twire  a  day,  each  reoeiving  at  a  meal  40  grarns  of  cooke<l  liver,  J  pint  of  milk, 
.V)  prraniM  of  lirciul,  and  :dl  the  water  it  wanted. 


456 


Wallace  Larkin  Chandler 


Dog  C  (Male  —  Weight  2.7  Kilograms) 

Date  of  blood  count 

(1917) 

Ked 

blood-cells 

(per  cubic 

millimeter) 

White 
blood-ceUs 
(per  cubic 
millimeter) 

Hemoglobin 

(per  cent) 

January  10 

5,196,000 
5.312,000 
5,344,000 
5,336,000 
5,040,000 

23,400 
25,000 
14,000 
15,000 
16,000 

60 

11 

60 

12   

65 

13   

60 

14           

64 

15               

16 

4,936,000 
5,178,000 
5,472,000 
5,496,000 
5,500,000 
5,608,000 
6,040,000 
§7,144,000 

11,600 
17,400 
16,600 
16,860 
17,200 
23,600 
19,620 
24,400 

62 

17 

18* 

60 
63 

19               

62 

20t. 

21 

22 

66 
62 

%72 

23 

80 

*  Dog  fumigated  for  five  hours  at  20°  C.  on  this  date. 

T  Symptoms  of  poisoning  appearing. 

%  The  comparison  standard  was  the  methemoglobin  standard,  so  that  if  methemoglobin  is  formed  within 
the  red  blood-cells,  as  Roth  claims,  and  these  cells  increase,  it  is  not  surprising  that  the  percentage  of 
hemoglobin  also  should  increase. 

§  Since  the  animal  refused  water  and  could  be  induced  to  take  only  a  very  little  milk,  it  is  probable  that 
the  rise  in  the  red-blood-eell  count  was  due  to  a  concentration  of  the  blood  resulting  from  lack  of  water. 

A  microscopic  examination  of  the  blood  of  this  dog  showed  that  the 
erythrocytes  were  sKghtly  distorted;  they  appeared  loose  and  sac4ike, 
and  would  not  form  rouleaux  in  fresh  mounts. 


Dog  D  (Male  —  Weight  2.5  Kilograms) 

Red 

White 

Date  of  blood  count 

blood-cells 

blood-cells 

Hemoglobin 

(1917) 

(per  cubic 
millimeter) 

(per  cubic 
millimeter) 

(per  cent) 

January  10 

4,904,000 
5,604,000 
5,296,000 
4,600,000 

15,720 
19,660 
14,660 
12,660 

65 

11   

65 

12* 

65 

13.. 

66 

14.  . 

5,446,000 
5,000,000 

14,060 
11,200 

66 

15.. 

64 

16.. 

5,040,000 

11,860 

62 

17.. 

18,. 
19.. 

4,798,000 
4,712,000 
5,024,000 

12,740 
11,660 
11,600 

62 
60 
58 

20.. 

4,974,000 

11,200 

85 

Dog  fumigated  for  four  hours  at  20°  C.  on  this  date. 


Physiological  Action  of  Nitrobenzene  Vapou  on  Animals    457 

This  animal  showed  a  slight  drop  in  the  red-cell  count  on  the  day 
following  the  fumigation,  but  (his  was  not  sufficiently  groat  to  be  of  any 
importance.  The  animal  d(!veloped  no  very  pi-onounccd  symptoms  of 
poisoning.  Dog  C,  on  the  other  hand,  did  develop  pronounced  symptoms, 
and  died  on  the  evening  of  January  23 ;  but  Dog  C  had  been  fumigated  for 
four  hours  at  18°  C.  on  December  12,  1916,  as  well  as  for  five  hours  in 
this  experiment.  As  the  result  of  the  first  fumigation,  however,  the  animal 
was  apparently  unharmed,  nor  was  there  any  change  in  the  blood  counts 
following  the  first  fumigation. 

SPECTROSCOPIC    EXAMINATION    OF    THE    BLOOD    OF    ANIMALS    POISONED    BY 

NITROBENZENE 

A  cat  was  fumigated  for  a  period  of  three  hours  at  a  temperature  of 
23.5°  C.  Shortly  after  the  fumigation  a  sample  of  blood  was  taken, 
diluterl  v/itli  distilled  water,  and  examined  spectroscopically.  When  the 
concentration  was  just  sufficient  to  cause  the  oxyhemoglobin  bands  to 
disappear,  a  distinct  band  appeared  between  C  and  D,  apparently  in  the 
exact  position  of  the  absorption  band  of  methemoglobin.  When  the 
sample  of  blood  was  sufficienth-  dilute  to  cause  the  oxyhemoglobin  bands 
to  stand  out  clearly,  the  absorption  band  between  C  and  D  disappeared 
or  was  very  faint.  The  undiluted  blood  was  coffee-colored,  and  the 
diluted  blood  had  the  appearance  of  methemoglobin  blood. 

A  young  dog,  a  cat,  a  i-abl^it,  a  guinea  pig,  a  chicken,  and  a  pigeon 
were  placed  together  in  the  fumigation  chamber  and  fumigated  for  a 
period  of  three  hours  at  22.5°  C.  At  the  end  of  the  fumigation,  a  sample 
of  blood  was  taken  from  each  and  examined  spectroscopically.  The  cat's 
blood  showed  the  above-described  band  faintly;  the  guinea  pig's  blood 
showed  the  band  very  distinctly;  the  samples  from  the  other  animals 
failed  to  show  the  band.  On  the  following  day,  blood  samples  were 
again  taken  and  examined.  The  sample  from  the  cat  showed  the  bantl 
more  markedly  than  on  the  preceding  day;  the  sample  from  the  guinea 
pig  did  not  show  the  band  at  all,  nor  did  the  samples  fiom  any  of  the 
other  animals. 

A  sample  of  blood  was  taken  from  a  healthy  cat  and  diluted  with  about 
fifty  volumes  of  distilled  water.     The  diluted  blood  was  then  shaken  in 


458  Wallace  Larkin  Chandler 

a  test  tube  with  a  few  drops  of  nitrobenzene.  When  examined  spectro- 
scopically,  the  sample  showed  no  trace  of  the  band,  but  only  oxyhemo- 
globin. The  sample  was'  allowed  to  stand  overnight,  but  the  band  even 
then  failed  to  appear.  Another  sample  was  taken,  diluted  as  before,  and 
shaken  with  nitrobenzene.  It  was  then  placed  in  an  incubator  at  a 
temperature  of  from  37°  to  38°  C  The  methemoglobin  band,  above 
described,  made  its  appearance  at  the  end  of  four  hours.  However, 
since  no  control  sample  was  incubated  at  the  same  time,  this  test  is  not 
a  positive  proof  that  nitrobenzene  can  form  methemoglobin  in  blood 
outside  of  the  animal. 

The  results  described  above  agree  fairly  well  with  the  findings  of 
Filehne  and  others.  There  has  been  some  disagreement  regarding  the 
nature  of  the  band  in  question;  however,  a  sample  of  methemoglobin 
prepared  in  the  laboratory  showed  an  absorption  band  in  exactly  the 
same  position  as  that  occupied  by  the  "  nitrobenzol  band." 

HISTOLOGICAL  EXAMINATION  OF  THE  TISSUES  OF  ANIMALS  POISONED  BY 

NITROBENZENE 

DOGS 

Four  half -grown  dogs,  all  from  the  same  litter,  were  kept  as  nearly 
as  possible  under  the  same  conditions.  On  January  24,  1917,  three  of 
them  (Dogs  E,  F,  and  G)  were  placed  together  in  the  fumigation  chamber 
and  fumigated  for  a  period  of  five  and  one-half  hours  at  a  temperature 
of  20°  C.  They  were  removed  from  the  tank  at  10  p.  m.,  apparently 
unharmed. 

On  January  25  Dog  F  showed  a  slight  lameness,  hid  himself  away 
in  a  dark  box,  and  did  not  eat  well.  The  other  two  animals  were  apparently 
normal. 

On  January  26  all  three  of  the  dogs  were  found  to  have  developed 
pronounced  symptoms  of  nitrobenzene  poisoning.  The  symptoms  were 
of  the  same  general  character  in  each,  but  were  more  advanced  in  Dog  F. 
All  the  animals  were  able  to  crawl  about;  they  had  a  partial' use  of  the 
fore  legs  but  were  not  able  to  use  the  muscles  of  the  hind  legs.  The 
knee-jerk  reflex  was  good  in  each  of  the  animals.  There  appeared  an 
incoordination  of  the  muscles  of  the  neck,  but  there  were  no  signs  of 


Physiological  Action  of  Nitrobenzene  Vapor  on  Animals    459 

nystagmus,  of  abnormal  pupil  reactions,  nor  that  the  animals  had  vomited. 
All  the  animals  drank  milk,  and  Dogs  E  and  G  ate  some  meat. 

On  January  27,  at  8  a.  m..  Dog  F  was  found  completely  paralyzed 
except  for  slight  movements  of  the  hind  legs,  and  there  was  a  copious 
secretion  of  saUva.  The  knee-jerk  reflex  was  good  in  both  legs.  At 
9.30  a.  m.  this  animal  was  howling  excitedly.  When  disturbed  it  went 
into  respiratory  convulsions  similar  to  those  described  for  Dog  VI  (page 
437).  The  animal  was  killed  at  noon  and  its  tissues  were  fixed  according 
to  the  methods  described  earlier  (page  429). 

Dog  E  refused  food  and  water  on  this  date,  was  wholly  helpless,  and 
howled  incessantly.  At  3  p.  m.,  three  hours  after  Dog  F  was  killed, 
this  animal  was  killed  and  its  tissues  were  fixed  in  the  manner 
described. 

Dog  G  took  milk  in  the  morning  on  this  date,  but  refused  it  at  night. 
It  exhibited  the  running  movements  of  the  legs  described  for  Dog  X 
(page  440),  and  howled  a  good  deal. 

On  January  28  intense  excitement  was  shown  by  Dog  G.  Its  legs  were 
moving  rapidly  with  the  running  movements  already  mentioned.  These 
movements  continued  for  long  periods  at  a  time,  and  then  the  leg  and 
the  abdominal  muscles  would  stiffen  in  violent  convulsions;  the  head 
thrashed  about  and  the  animal  gave  guttural  sounds  as  if  worrying  a  rat ; 
finally  the  animal  would  grasp  part  of  its  bedding  with  its  teeth  and  hold 
it  firmly  for  a  moment,  during  which  time  respiration  would  cease,  and 
then,  after  a  few  gasps,  the  running  movements  would  begin  again.  At 
times  the  animal  shook  its  head  vigorously,  as  if  trjdng  to  get  rid  of 
something  in  its  ear.  This  dog  and  the  control,  Dog  H,  were  killed  in 
the  course  of  the  morning  and  their  tissues  were  fixed,  as  nearly  as  possible, 
after  the  same  manner  as  were  the  tissues  of  Dogs  E  and  F. 

Corresponding  pieces  of  tissue  from  the  different  levels  of  the  central 
nervous  system  of  each  of  the  four  animals  were  carried  thru  the  same 
fluids,  and  were  finally  stained  and  mounted  on  the  same  slide,  as  described 
on  page  430. 

No  histological  changes  could  l)e  observcnl  in  th(;  ccjlls  of  any  part  of 
the  central  nervous  system,  except  in  the  Purkinje  cells  of  the  cerebellum. 
The  changes  in  these  cells  were  typically  chromatolytic  degenerations. 


460  Wallace  Lakkin  Chandlek 


Plate  VI 

1-4,  Photomicrographs  of  Purkinje  cells  from  the  cerebella  of  four  dogs  of  the  same 
litter.     X  525 

1,  Normal  cells  from  control  animal.  The  presence  of  tigroid  bodies  (Nissl  bodies  [N] ) 
is  to  be  noted 

2,  Cell  from  animal  poisoned  by  the  vapor  of  nitrobenzene,  showing  the  first  stages  of 
chromatolytic  degeneration.  The  much  swollen  cell-body,  and  the  absence  of  tigroid  bodies 
except  a  few  in  the  vicinity  of  the  nucleus,  are  apparent 

3,  Cells  from  animal  poisoned  by  the  vapor  of  nitrobenzene,  showing  (a)  the  swollen 
cell-body  and  the  absence  of  tigroid  bodies,  and  (b)  a  homogeneous  appearance  of  the  cyto- 
plasm and  the  absence  of  tigroid  bodies 

4,  Remains  of  a  Purkinje  cell  which  has  undergone  chromatolytic  degeneration.  (From 
the  cerebellum  of  a  dog  poisoned  by  the  vapor  of  nitrobenzene) 

5-6,  Photomicrographs  of  motor  cells  from  the  ventral  horn  of  the  cervical  cords  of  two 
dogs  from  the  same  litter,     x  525 

5,  Normal  cell  from  control  animal 

6,  Cell,  apparently  normal,  from  animal  (Dog  F)  poisoned  by  the  vapor  of  nitrobenzene 

7-8,  Photomicrographs  of  sections  thru  similar  convolutions  of  the  cerebeUa  of  two  dogs. 
x60 

7,  From  control  animal,  showing  the  presence  of  Purkinje  cells  (P)  and  a  normal  cortical 
area  (c) 

8,  From  animal  killed  nine  months  after  being  poisoned  by  the  vapor  of  nitrobenzene 
(Dog  XVIII) ,  showing  the  absence  of  Purkinje  cells  and  a  much  atrophied  cortical  area 


Memoir  20 


Plate  VI 


PiivsK)LO(ii('AL  Action  of  Nitrobenzene  Vafoh  on  Animals     461 

The  Puikinje  cells  from  Dog  F  (Plate  VI,  2)  were  greatly  swollen,  and 
the  Nissl  bodies  (tigroid  bodies)  were  almost  entirely  absent.  There  was 
no  stainable  substance  near  the  periphery,  and  that  around  the  nucleus 
was  massed  and  indefinite.  Some  of  the  Purkinje  cells  from  Dog  E 
were  like  those  from  Dog  F;  others  were  very  much  shrunken,  about 
one-half  "normal"  size,  and  the  whole  was  a  darkly  stained,  indefinite 
mass  with  the  nucleus  almost  obhterated.  The  Purkinje  cells  from  Dog  G 
were  all  veiy  much  shrunken  (Plate  VI,  4),  and  no  Nissl  bodies  were  to 
be  seen.  There  was  a  small  amount  of  stainable  substance,  massed  and 
clinging  about  a  much-shrunken  nucleus.  In  many  cases  the  nucleus 
had  disappeared,  and  in  numerous  instances  the  entire  cell  seems  to  have 
disappeared.  The  Purkinje  cells  from  the  control  animal,  Dog  H,  were 
all  apparently  normal,  the  tigroid  bodies  staining  excellently  (Plate  VI,  1). 

The  above  experiment  was  repeated,  and  this  time  two  controls  were 
used  instead  of  one.  One  of  the  poisoned  animals  was  killed  shortly 
after  the  first  appearance  of  the  symptoms,  and  only  a  few  of  the  Purkinje 
cells  from  this  animal  showed  degeneration.  Another  one  of  the  animals 
was  killed  before  the  symptoms  were  very  far  advanced,  and  in  this  case 
but  few  of  the  Purkinje  cells  looked  wholly  normal  and  some  of  them 
showed  definite  chromatolytic  degeneration.  The  third  poisoned  animal 
was  killed  after  the  symptoms  were  well  advanced,  and  the  cells  from  this 
animal  had  become  so  thoroly  degenerated  that  there  was  scarcely  any- 
thing left  of  them  but  irregular  blotches.  The  Purkinje  cells  from  the 
control  animals  were  all  apparently  normal. 

Sections  were  made,  stained,  and  mounted  on  the  same  slide,  of  the 
following  additional  tissues  from  the  control  animals  and  from  the  animal 
in  this  later  experiment  in  which  the  symptoms  of  poisoning  were  well 
advanced:  liver,  spleen,  thyroid,  adrenal  gland,  duodenum,  and  rectus 
femoris  muscle.  No  signs  of  degeneration  could  be  observed  in  any 
of  these  tissues,  but  there  appeared  to  be  a  slight  hyperemia  of  the  fiver, 
the  duodenum,  and  the  rectus  femoris  muscle;  these  tissues  were  dissected 
out  before  the  infiltration  of  the  saline  solution,  so  that  the  blood  remained 
in  them. 

BIRDS 

Two  pigeons  were  fumigated  with  nitrobenzene,  and  when  the  symptoms 
lx;came  pronounced,  they,  togethiM-  with  a  control,  w(!re  killed  by  clipping 


462  Wallace  Larkin  Chandler 

off  their  heads.  The  cerebellum  was  hurriedly  dissected  out  and  small 
sections  from  different  parts  were  placed  directly  into  the  fixing  fluid. 
The  remainder  of  the  technique  was  the  same  as  that  employed  in  the 
case  of  the  dogs.  The  same  degenerative  changes  in  the  Purkinje  cells 
were  found  as  were  found  in  the  case  of  the  dogs.  The  Purkinje  cells 
from  the  control  bird  were  normal. 

Two  chickens,  poisoned  and  killed  in  the  same  manner,  showed  the 
same  type  of  degeneration  in  the  Purkinje  cells  as  is  described  above, 
while  the  cells  from  the  control  bird,  stained  and  mounted  on  the  same 
slide  with  those  from  the  poisoned  bird,  were  apparently  normal. 

THE  SYMPTOM  COMPLEX  OF  NITROBENZENE  POISONING 

Unlike  Letheby  and  Filehne,  the  writer  has  been  unable  to  divide 
the  symptom  complex  of  nitrobenzene  poisoning  into  two  types  —  that 
accompanying  a  rapid  action  of  the  drug,  and  that  accompanying  a  I'etarded 
action.  As  a  matter  of  fact,  the  symptoms  are  never  sufficiently  uniform 
in  any  case,  whether  the  action  is  slow  or  rapid,  to  permit  of  classification 
into  types.  There  is  a  probability,  however,  that  the  symptom  complex 
may  be  somewhat  different  in  widely  separated  groups  of  animals;  but 
this  can  be  determined  only  by  a  summation  of  all  the  symptoms  observed 
in  experiments  on  a  large  number  of  individuals  from  each  group. 

So  far  as  has  been  observed  by  the  writer,  the  dominant  symptom  in- 
frogs  and  in  insects  is  a  general  depression,  tho  in  insects  a  tremulous 
movement  of  the  legs  has  also  been  observed.  In  birds  and  mammals, 
one  or  more  or  all  of  the  following  symptoms  may  appear  in  acute  cases 
of  poisoning  by  the  vapor  of  nitrobenzene:  cyanosis,  nausea,  vomiting, 
ataxia,  asynergia  (distinguished  from  other  types  of  cerebellar  ataxia 
in  that  there  appears  a  retro-  or  propulsion  in  an  antero-posterior  plane 
instead  of  a  lateral,  the  legs  appearing  as  if  either  running  away  from  the 
body  and  throwing  the  animal  backward,  or  failing  to  keep  up  with  the 
body  and  throwing  the  animal  on  its  head),  impairment  of  digestive 
functions,  nystagmus,  equal  or  unequal  dilation  or  contraction  of  the 
pupils,  irritability,  tenderness  of  the  occiput  (headache),  unconsciousness, 
hallucinations,  adiadochokinesis,  slowing  of  the  pulse  rate,  irregular  and 
weakened  respiration,  palpitation  of  the  muscles,  rapid  (running)  move- 
ments of  the  legs,  rotation  of  the  head  and  neck  describing  broad  circles, 
rotation  of  the  body  around  its  longitudinal  axis,  asthenia,  and  the  nitro- 


Physiological  Action  of  Nitrobenzene  Vapor  on  Animals     463 

benzene  l)i-eath-.  In  chronic  cases  the  symptoms  which  persist  are: 
asthenia,  ataxia,  and  (described  in  man)  anemia,  malnutrition,  and 
Korsakoff's  psychosis. 

interpretation  of  the  symptoms  named 

While  there  is  scarcely  a  single  one  of  the  symptoms  described  above 
that  may  not  be  referred  to  disorders  of  the  central  nervous  system, 
still  there  is  no  doubt  that  nitrobenzene  exerts  a  more  or  less  serious 
local  action  on  other  tissues.  The  cyanosis  observed  in  most  cases  of 
acute  poisoning  is  undoubtedly  due  to  a  direct  action  of  nitrobenzene 
on  the  blood;  the  blood  has  a  dark  brown  color,  and  the  presence  of 
methemoglobin  is  demonstrable,  at  least  in  some  cases,  by  spectroscopic 
analysis.  Just  how  the  changes  in  the  blood  are  brought  about  is  not  defi- 
nitety  understood.  Roth  (1913)  thinks  that  the  nitrobenzene  is  converted 
into  paraminophenol  and  that  the  latter  drug  acts  on  the  red  blood-cells, 
forming  intracellular  methemoglobin;  he  found  no  methemoglobin  in  the 
serum  of  certrifugalized  blood.  Tiirk  (cited  by  Adams,  1912)  says  that 
not  only  is  there  a  forming  of  methemoglobin,  but  there  is  also  a 
destruction  of  the  erythrocytes,  due,  he  thinks,  either  to  intravascular 
hemotysis  or  to  a  hyperfunctioning  of  the  blood-destroying  organs.  That 
methemoglobin  is  formed,  in  certain  cases,  was  demonstrated  by  the 
writer's  experiments;  the  writer  was  unable,  however,  to  demonstrate  the 
destruction  of  erythrocytes,  altho  some  morphological  alterations  of  these 
cells  were  apparent.  Also,  Filehne  (1878)  has  shown  that  nitrobenzene 
has  a  direct  action  on  the  muscle  substance,  causing  the  muscle  to  contract 
in  rigor  mortis;  even  the  heart  muscle  was  affected,  according  to  him. 
Certain  it  is  that  nitrobenzene  has  an  irritating  action  on  the  tongue 
and  the  mucosae;  and,  since  it  passes  unchanged  readily  from  the  blood 
to  other  tissues,  it  is  not  impossible  that  it  may  have  an  irritating  action 
on  the  deeper  tissues  also.  However,  if  the  symptoms  produced  are  due 
to  a  direct  action  of  the  drug  upon  the  blood,  why,  then,  should  there 
be  such  a  long  latent  period  in  most  cases?  Furthermore,  if  the  symptoms 
are  due  to  a  dii'ect  action  of  the  drug  upon  muscles,  glands,  or  abdominal 
organs,  then  these  tissues  should  show  histological  changes;  but  the 
writer  has  failed  to  find  anything  more  severe  than  a  slight  hyperemia, 
the  cause  of  which  may  be  easily  explained  on  the  basis  of  the  hyper- 
activity of  th(^  organs  concerned. 


464  Wallace  Larkin  Chandler 

Disturbance  of  digestive  f  mictions 

The  retardation,  or  in  some  cases  even  the  cessation,  of  digestive  proc- 
esses in  the  poisoned  animals  is  not  wholly  understood.  Casper  (1859) 
observed  that  in  post-mortem  examinations  of  animals  poisoned  by- 
nitrobenzene  the  stomach  contents  were  always  alkaline;  and,  since  the 
acidity  of  the  fluid  in  the  stomach,  especially  in  the  pyloric  end,  has  been 
shown  to  be  essential  to  gastric  digestion  (Howell,  1918),  it  is  possible 
that  nitrobenzene  in  some  way  hinders  the  formation  or  secretion  of 
hydrochloric  acid. 

Cerebellar  disturbances 

Turning  now  to  the  evidence  indicating  cerebellar  involvement,  the 
following  facts  may  be  noted.  From  the  descriptions  it  will  be  seen,  as 
already  stated,  that  there  is  scarcely  a  single  one  of  the  symptoms  appear- 
ing in  cases  of  nitrobenzene  poisoning  that  cannot  be  referred  to  disturb- 
ances in  the  cerebellum  or  the  cerebellar  paths,  barring  those  which  are 
undoubtedly  due  to  a  direct  action  on  the  blood.  Moreover,  this  con- 
clusion appears  to  be  borne  out  by  histological  data,  since  the 
cerebellum  is  the  only  organ  in  which  definite  histological  changes  were 
found. 

Nausea  and  vomiting. —  Nausea  and  vomiting,  accompanied  by  some 
of  the  symptoms  named  above,  may  be  due  to  disorders  of  the  cerebellar 
paths  (Jelliffe,  1913).  Nausea  and  vomiting  may  appear  during  the 
fumigation  process,  altho  the  animal  may  show  no  other  pronounced 
symptoms  for  several  days;  but  this  does  not  preclude  the  possibility  that 
nausea  and  vomiting  are  the  result  of  the  action  of  nitrobenzene  on  the 
cerebellum,  since  it  is  possible  that  a  sufficient  amount  of  the  drug  may  be 
concentrated  in  the  cerebellum  to  cause  these  first  sj^mptoms  without 
enough  being  present  to  produce  any  further  symptoms;  moreover,  if  the 
animal  were  left  in  the  fumigation  chamber  for  a  little  longer  period, 
the  other  symptoms  would  appear  very  quickly.  If  the  cause  of  the 
vomiting  were  to  be.  ascribed  to  a  peripheral  action  of  the  drug,  then  the 
same  explanation  would  have  to  be  given  as  for  the  cause  of  the  vomiting 
after  the  latent  period,  it  would  seem. 

Ataxia.—  The  type  of  motor  ataxia  exhibited  by  animals  poisoned 
by  nitrobenzene,   especially  by  dogs  and  birds,  is  typically  cerebellar. 


Physiological  Action  of  Nitrobenzene  Vapor  on  Animals    465 

This  is  indicated  by  tho  hohhlc  or  spiawlino-  jrait  in  walking,  the  position 
of  the  legs  in  stantling,  and  other  characteristics  mcnitioncd  heretofore 
(page  462). 

Adindochohinesk. —  Adiad()clu)kin(>sis  is,  jierliaps,  the  best  interpretation 
of  the  type  of  convulsions  observed.  This  is  a  term  originally  used  by 
Babinski  to  describe  a  pecuhar  type  of  incoordination  in  patients  (Jelhffe, 
1913).  It  is  seen  in  the  absence  of  paralysis,  muscle  palsies,  and  sensibility 
disturbances,  and  is  characterized  by  a  loss  of  ability  to  carry  out  rapidly 
alternating  movements,  such  as  flexion  and  extension  of  the  forearm  on 
the  arm.  In  dogs,  })irds,  rats,  and  other  animals  poisoned  by  nitro- 
benzene, it  was  observed  that  at  times  one  set  of  muscles  would.be  con- 
tracted and  another  set  relaxt^d,  and  at  other  times  just  the  reverse 
happened.  For  example,  if  motor  impulses  passed  to  the  extensors  of 
the  hind  legs,  the  animal  was  unable  to  stop  these  impulses  or  to  use 
the  antagonistic  muscles,  and  the  leg  remained  for  a  time  rigidly  extended ; 
again,  if  the  impulses  passed  to  the  flexors,  then  the  animal  was  unable 
to  extend  the  leg.  This  condition,  according  to  Babinski,  indicates 
involvement  of  the  cerebellar  paths. 

Nystagmus. —  According  to  JelHffe  (1913),  Holmes  states  that 
"  nystagmus  .is  a  common  and  very  valuable  localizing  sign  of  local  cerebellar 
lesions.  It  is  almost  certainly  a  true  cerebellar  symptom."  Nystagmus 
does  not  always  appear,  ]mt  when  present  it  is  invariably  a  cerebellar 
type,  distinguishable  from  vestibular  nystagmus  by  the  jerky  movements 
of  the  eyeball.  The  shifting  is  in  a  lateral  plane,  and  is  equally  rapi'd 
in  either  direction. 

Asthenia. —  Asthenia  is  one  of  the  first  symptoms  to  appear  and  is 
almost  invariably  present.  The  animal  is  usually  so  weak  that  when  it 
is  placed  on  its  feet,  its  legs  literally  double  up  beneath  it,  and  its  head 
sways  about  as  if-  the  neck  were  disjointed.  This  symptom,  according  to 
Jelliffe  (1913),  indicates  "disorder  of  the  tractus  cerebellovestibularis 
spinalis,  or  rubro-spinalis." 

Hypotonus. —  Hypotonus,  as  revealed  by  palpation  of  the  muscles  and 
Vjy  special  tests  of  the  tendon  reflexes,  was  observed  in  the  more  advanced 
cases,  especially,  of  nitrobenzene  poisoning.  In  some  cases  this  symptom 
was  accompanifid  by  poor  tendon  reflex  reactions,  while  in  other  cases 
the  tendon  reflexes  were  noiinul  oi'  even  exaggerated.  These  conditions 
indicate  cerebellar  hypotonus. 


466  Wallace  Larkin  Chandler 

Disturbance  of  respiration. —  In  moribund  animals  poisoned  by  nitro- 
benzene, one  almost  invariably  observes  a  disturbance  of  respiration,  and 
in  some  instances  a  slowing  of  the  pulse  rate.  Musser  (1904)  states  that 
cerebellar  tumors  often  cause  symptoms  of  this  type. 

Headache  and  tenderness  of  the  occiput. —  Headache  in  animals  is  very 
difficult,  if  not  entirely  impossible,  to  determine.  However,  certain  actions 
exhibited  by  animals  poisoned  by  nitrobenzene  might  be  interpreted  to 
indicate  headache;  for  instance,  the  animal's  desire  to  press  its  head  against 
some  object.  Dog  I  was  observed  to  stand  for  long  intervals  with  its 
head  pressed  against  the  attendant's  legs  or  against  some  solid  object 
(page  431).  And  certainly  the  fact  that  the  animal  exhibited  some  dis- 
comfort if  the  back  of  its  head  was  touched,  indicated  a  tenderness  in  the 
vicinity  of  the  occiput.  These  symptoms  have  been  observed  in  cases 
of  cerebellar  tumors. 

Circus  movements. —  Rotation  of  the  head  and  the  neck  was  always 
observed  in  the  case  of  birds  poisoned  by  nitrobenzene.  This  is  a  character- 
istic symptom  observed  in  cerebellar  pigeons.  Rotation  of  the  body  about  its 
longitudinal  axis,  observed  especially  in  gray  rats  and  sometimes  in  dogs, 
is  a  symptom  exhibited  in  cerebellar  mammals. 

CAUSE  OF  THE  LATENT  PERIOD 

The  fact  that  there  often  exists  a  long  latent  period,  the  period  of  time 
elapsing  between  the  administration  of  the  drug  and  the  onset  of  the 
symptoms,  has  led  to  much  theorizing  as  to  its  cause.  The  following  two 
theories  have  been  the  most  popular:  the  theory  advanced  by  Olhvier 
and  Bergeron  and  held  to  by  Letheby,  that  nitrobenzene  is  converted 
into  anilin  in  the  body  and  that  time  is  required  for  this  transformation;, 
and  the  theory  investigated  first  by  Filehne  and  accepted  by  most  recent 
writers,  that  the  drug  is  so  lightly  soluble  in  the  tissues  of  the  body  that 
time  is  required  for  the  absorption  of  it  in  sufficient  amounts  to  produce 
the  poisonous  effects. 

Filehne  has  ably  shown  that  the  action  of  the  drug  does  not  depend  on 
its  conversion  into  other  chemicals.  Furthermore,  he  failed  to  find  any 
trace  of  anilin  in  any  of  the  organs  of  animals  poisoned  by  nitrobenzene 
even  by  the  hypochlorite  test.  His  criticism  of  the  isophenylcyanide  test 
(used  by  Letheby)  is  justified,  since  in  conducting  this  test  nitrobenzene 


Physiological  Action  of  Nitrobenzene  Vapor  on  Animals     407 

is  itself  convci'tcd  into  auilin.  Filehne  has  shown  also  that  the  theor}- 
of  slow  absorption  does  not  account  for  the  rapid  action  of  the  ch'ug 
observed  in  certain  cases. 

In  all  probability  the  rate  of  absorption  of  nitrobenzene  by  the  body 
tissues  does  have  something  to  do  with  the  cause  of  the  latent  period; 
but  the  explanation  must  be  primarily  based  on  the  readiness  with  which 
the  drug  is  absorbed  by  certain  tissues  as  compared  with  other  tissues, 
and  not  alone  on  the  rate  of  absorption  by  the  tissues  in  general.  It  will 
be  recalled  that  nitrobenzene  is  readily  soluble  in  oils  and  the  liquid  fats; 
it  is  soluble  also  to  a  certain  extent  in  lipoids,  but  probably  to  a  less  extent 
in  certain  lipoids  than  in  others,  and  even  the  same  lipoid  may  absorb 
the  drug  more  readily  under  certain  conditions  than  under  other  con- 
ditions. 

When  the  drug  is  administered  by  vapor  inhalation,  the  amount 
absorbed  by  the  blood  (at  a  given  vapor  pressure)  depends,  undoubtedly, 
on  the  amount  and  the  condition  of  the  fats  or  the  lipoids  in  the  blood. 
Also,  the  amount  absorbed  from  the  blood  and  held  in  solution  by  the 
body  fats  is,  in  all  probability,  directly  proportional  to  the  absorption 
power  of  the  body  fats  over  the  absorption  power  of  the  fats  in  the  blood. 

Since  nitrobenzene  is  more  readily  absorbed  by  the  liquid  body-fats 
than  by  the  lipoids  (of  the  lirain),  large  amounts  of  it  may  be  stored  in 
the  liquid  fats  of  the  body  without  the  animal's  showing  any  immediate 
symptoms  of  poisoning.  Moreover,  since  the  action  of  the  drug  on  the 
cells  of  the  brain  probably  depends  on  its  concentration  in  the  vicinity  of 
these  cells  —  as  in  the  case  of  chloroform,  ether,  and  other  drugs  that 
act  directly  on  the  cells  of  the  brain  —  niti'obenzcne,  depending  on  the 
amount  and  the  condition  of  the  lipoids  and  the  fats  held'  in  suspension 
in  the  blood,  may  be  picked  up  from  the  body  fats  by  the  blood  in  such 
small  amounts  as  to  be  in  time  entirely  eliminated  from  the  body  without 
ever  giving  a  sufficient  concentration  in  the  vicinity  of  Pui'kinje's  cells 
to  cause  .symptoms  of  poisoning;  so  that  large  amounts  of  the  drug  may 
be  stored  in  the  body  without  the  animal's  ever  showing  any  symptom 
of  poisoning. 

On  the  other  hand,  depending  on  the  concentration  of  nitrobenzene  in 
the  blood  supply  to  the  cerc^bellum,  a  sufficient  concentration  of  the  drug 
in  the  vicinity  of  I  he  Purkinje  cells  might  be  reached,  even  after  miniite 


468  Wallace  Larkin  Chandler 

doses,  to  affect  these  cells  and  produce  the  typical  symptoms  of  poisoning, 
or  even  death,  within  a  very  short  time  after  the  administering  of  the 
poison. 

If  the  above  assumptions  are  correct  —  and  they  can  be  so  proved  only 
after  a  long  series  of  experiments  dealing  more  exhaustively  with  the 
physics,  chemistry,  and  physiology  of  the  subject  — -  then  it  is  not  sur- 
prising that,  as  was  found,  the  latent  period,  as  well  as  the  intensity  of 
the  action  of  the  drug,  should  vary  in  different  individual  animals,  or 
even  in  the  same  individual  at  different  periods  of  time;  for  neither  the 
amount  nor  the  kind  of  fats  in  the  blood  or  the  nervous  tissues  is  abso- 
lutely constant. 

CONCLUSIONS 

The  present  work  has  confirmed  the  findings  of  previous  investigators 
regarding  all  six  of  the  points  listed  in  the  first  paragraph  following  the 
review  of  the  literature.  In  addition  the  following  conclusions  have  been 
deduced : 

1.  Aside  from  the  possible  disturbance  of  digestive  functions  and  a 
possible  asphyxia  due  to  a  direct  action  of  nitrobenzene  on  the  blood, 
most,  if  not  all,  of  the  observed  symptoms  of  nitrobenzene  poisoning  may 
be  explained  on  the  basis  of  disturbances  in  the  cerebellum  or  the  cerebellar 
paths. 

2.  Toxic  doses  of  nitrobenzene,  when  administered  by  vapor  inhalation, 
exert  a  direct  action  on  the  Purkinje  cells  in  the  cerebellum,  causing 
chromatolytic  degeneration  of  these  cells. 

3.  Histological  examinations  have  failed  to  reveal  any  definite  changes 
in  any  of  the  organs  of  the  body  except  the  blood  (presence  of  methemo- 
globin  and  morphological  alterations  of  erythrocytes)  and  the  cerebellum 
(chromatolytic  degeneration  of  the  Purkinje  cells) .^^ 

4.  The  size  of  the  lethal  dose  probably  depends  on  conditions  such  as 
the  amount  and  the  kind  of  fats  in  the  blood,  which  favor  or  disfavor  a 
concentration  of  the  drug  in  the  vicinity  of  the  nerve  cells. 

5.  The  latent  period  (the  time  elapsing  between  the  administration  of 
the  poison  and  the  onset  of  the  symptoms)  is  undoubtedly  due  to  the 

"  The  writer  does  not  mean  to  assert  that  histological  changes  do  not  occur  in  other  tissues,  especially 
in  other  parts  of  the  central  nervous  system.  Indeea,  a  further  study  of  tlie  present  sections  may  yet 
reveal  such  changes.  It  would  be  strange  if  the  action  of  nitrobenzene  on  the  central  nervous  system  were 
confined  to  a  single  type  of  cells  only.  Probably  in  cases  of  fatal  poisoning  other  nerve  cells  are  irtvolved 
also,  but  it  will  be  difficult  to  determine  whether  such  changes  are  due  to  a  direct  action  of  the  poison  or 
are  the  result  of  a  complication  of  changes  attending  death  by  poisoning.  Further  investigations  are  being 
made  along  this  line. 


Physiological  Action  of  Nitrobenzene  ^apok  on  Animals     469 

absorption  of  the  nitrobenzene  from  the  blood,  and  its  retention  by  the 
liquid  fats  of  the  bod}^  in  which  it  is  easily  soluble.  As  the  concentration 
of  the  poison  in  the  blood  lipoids  and  fats  diminishes,  in  "relation  to  its 
concentration  in  the  body  fats,  the  nitrobenzene  is  given  up  again  to  the 
blood;  and  in  the  course  of  time,  a  sufficient  concent ratioji  of  the  poison 
in  the  lipoids  of  the  cerebellar  or  other  brain  cells  is  reached  to  produce 
an  onset  of  the  motor  symptoms.  The  time  requircnl  to  bring  this  about 
(the  latent  period)  depends  on  the  same  factors  as  those  on  which  the  size 
of  the  lethal  dose  depends,  and  is  undoubtedly  hastened  by  the  ingestion 
of  solvents  of  nitrobenzene,  such  as  alcohols,  fats,  oils,  milk,  and  the  like. 
Possibly,  also,  the  condition  of  the  brain  lipoids  at  a  given  time  may  be 
an  important  factor  in  hastening  or  retarding  the  al)sorption  of  nitroben- 
zene by  these  lipoids. 

6.  Nitrobenzene  cannot  be  used,  with  au}^  degree  of  safety,  for  the 
fumigation  of  animals  to  destroy  their  external  parasites.  The  lethal 
dose  for  })irds  and  manunals  is  i-ather  variable  but  it  may  be  very  small; 
and  from  these  experiments  it  will  be  seen  that  apparently  a  shoi'ter  period 
of  fumigation  at  a  given  temperature  is  required  in  older  to  kill  a  domestic 
animal  than  to  kill  even  a  fair  proportion  of  eith(M-  fleas  or  biting  lice. 
However,  as  has  been  pointed  out  by  the  writer  in  a  previous  paper 
(Chandler,  1917),  the  drug  may  be  used,  with  the  exercise  of  caution,  for 
collecting  external  parasites  from  animals,  by  fumigating  the  animals  at 
low  temperatures  and  for  shoit  periods  of  time  —  at  a  temperature  not 
over  20°  C,  and  for  a  period  not  longer  than  one  and  one-half  hours. 
Under  these  conditions  the  i)arasites  may  be  stupefied  without  any  appre- 
ciable damage  being  done  to  t  he  host ;  but  the  drug  is  dangerous  to  handle 
under  any  conditions. 

7.  Because  of  the  extreme  toxic  properties  and  the  su])tle  action  of 
nitrobenzene,  the  following  uses  of  this  drug  should  be  prohibited  by 
legislation :  for  perfuming  soaps,  lotions,  and  pomades ;  as  a  solvent  in  shoe 
polish,  floor  wax,  and  th(>  like;  and  especially  .as  an  ingredient  of  flavoring 
extracts,  confections,  and  liqueurs.  The  drug  should  be  regarded  as  one 
of  the  most  dangerous  of  poisons,  and  its  sale  and  use  should  be  regulated 
by  law  just  as  in  th(>  cas(;  of  any  other  deadly  poison. 

8.  Nitrobenzene  should  be  given  serious  consideration  as  an  industrial 
poison.     Munition  plants,  dye  works,  and  other  factories  which  handle 


470  Wallace  Larkin  Chandler 

nitrobenzene,  should  be  inspected  with  the  view  of  instalHng  devices  to 
prevent  workmen  from  inhahng  the  vapor  or  coming  into  contact  with 
the  hquid. 

9.  In  view  of  the  evident  relation  existing  between  the  lethal  dose  of 
nitrobenzene  and  the  amount  and  kind  of  fats  and  other  solvents  of  the 
drug  present  in  the  blood,  it  would  appear  highly  desirable  that  further 
investigations  should  be  undertaken  with  the  view  of  working  out  rational 
therapeutics  for  cases  of  poisoning. 

10.  Since  the  findings  in  these  experiments  indicate  that  the  symptoms 
of  nitrobenzene  poisoning  are  caused  by  a  direct  action  of  the  drug  on  the 
Purkinje  cells,  causing  these  cells  to  degenerate,  and  since  different  sets 
of  muscles  appear  to  be  involved  at  different  times,  it  would  appear  that 
further  investigations  might  be  of  value  from  the  standpoint  of  obtaining 
more  exact  data  regarding  the  localization  of  functions  in  the  cerebellum. 


1 


Physiological  Action  of  Nitrobenzene  Vapor  on  Animals     471 


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Memoir  18,  A  Study  of  Bacteria  in  Ice  Cream  during  Storarje,  the  second  preceding  number  in  this  series 
of  pubhcations,  was  mailed  on  February  12,  1919. 


Date  Due 

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Physiological  action  of  nitroben- 
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